Conference Agenda

Several bioenergetic processes take place in membrane bound complexes that catalyze intricate proton and electron reactions which generate a proton motive force needed for function. Ionizable groups generate proton gradients but are often buried in hydrophobic pockets inside the membrane. Estimation of pK_a values of these key residues is of great importance to describe the dynamics of the system and additionally determine proton pathways. Despite major structural, biochemical and computational advances, pK_a values of these complexes remain poorly understood and are a major challenge for modern life sciences [1]. While there are many tools to predict pK_a values in soluble proteins based either on physical or chemical properties [2], as well as database learning tools with good accuracy [3], membrane bound pK_a values remain a challenge. Currently there is no larger database available for pK_a values of buried titratable groups due to its complexity, which hinders the development of more reliable, yet typically quick prediction methods. In addition, rare events like wetting-dewetting transitions, which cannot be inferred from e.g. crystal- or cryo-EM structure or standard MD simulations alone, are major factors that are missing from the current picture.

In this work we review the current state of PBE/MC methodology and available benchmark data to be able to predict pK_a values in soluble proteins with similar accuracy, and based on the lessons learned we develop a size-consistent approach to predict pK_a values in membrane bound proteins.

[1] Sorenson, J. L.; Mercedes, R.; Schlessman, J. L.; García-Moreno, B., PKA Values of Buried Groups in Proteins are Sensitive to the Global Thermodynamic Stability. Biophys. J. 2015, 108, 530a-531a.

[2] Reis, P. B. P. S.; Vila-Viçosa, D.; Rocchia, W.; Machuqueiro, M., PypKa: A Flexible Python Module for Poisson–Boltzmann-Based pKa Calculations. J. Chem. Inf. Model. 2020, 60, 4442-4448.

[3] Chen, A. Y.; Lee, J.; Damjanovic, A.; Brooks, B. R., Protein pK_a Prediction by Tree-Based Machine Learning. J. Chem. Comput. Theo. 2022, 18, 2673-2686.

Complex I is a redox-driven proton pump that couples electron transport with proton pumping across a biological membrane, powering oxidative phosphorylation and synthesis of ATP. Despite significant advances, the long-range proton pumping mechanism is still unsolved and much debated, with recent work challenging the subunits involved in pumping across the membrane. Here we combine computationally-guided protein engineering and site-directed mutagenesis with biophysical pumping experiments and ¹⁹F-NMR to derive a key understanding of the proton translocation mechanism in the membrane module of Complex I. We identify central gating residues and conformational changes that establish the tight electron-proton coupling in Complex I, and we further show how disease-related mutations perturb the proton transport activity. Our combined findings provide new functional insight into the long-range energy transduction mechanism of Complex I, and a blueprint for understand principles underlying mitochondrial diseases.

1] Kaila, V. R. I.; Wikström, M. Architecture of bacterial respiratory chains. Nat. Rev. Microbiol. (2021), 19, 319−330.

[2] Mitchell, P. Coupling of Phosphorylation to Electron and Hydrogen Transfer by a Chemi-Osmotic Type of Mechanism. Nature (1961), 191, 144−148.

[3] Ville R I Kaila. Long-range proton-coupled electron transfer in biological energy conversion: towards mechanistic understanding of respiratory complex I. J R Soc Interface, 15(141), April 2018.

[4] Beghiah A et al. (in Review)

F_OF₁-ATPase is the key enzyme in Escherichia coli under fermentative conditions pumping out protons to the external environment [1]. Here, the role of proton ATPase in the interplay between H⁺/H₂ cycles and the formation of proton motive force (Δp) was investigated depending on the main fermenting substrate in the mixture (glucose, glycerol and formate) at pH 7.5. E. coli MC4100 wild type (wt) and DK8 (lacking F_OF₁-ATPase) mutant were used. The lack of F_OF₁ showed a change in the metabolic pathways, resulting in a reduction of the redox state and causing variation in pH_in and pH_ex. Thus, the transmembrane proton gradient (ΔpH) was increased by ~0.24 units in the DK8 strain during glucose fermentation (20 h), compared to wt. At the same time, membrane potential (ΔΨ) was decreased in the DK8 indicating that F_OF₁ generates ΔΨ by ~32 mV. As a result, Δp was decreased by ~17mV. In contrast, a simultaneous decrease of ΔpH and ΔΨ was detected during glycerol utilization (72 h), which caused the Δp variation by ~47 mV. This phenomenon was due to the increased contribution of F_OF₁ in the rate of H⁺ flux at 72 h. Furthermore, decreased proton conductance in the F_OF₁-lacking cells demonstrated efficient energy transduction to save viability when the interaction between H⁺/H₂ cycles was absent. Moreover, the lack of F_OF₁ decreased hydrogenase (Hyd) and formate dehydrogenase H (FDH-H) activity during glycerol utilization and only Hyd activity during glucose fermentation. These results suggest the proton transfer via Fdh- H -> F_OF₁ <-> Hyd pathway during glucose utilization.

Taken together, F_OF₁-ATPase had a contribution to the formation of Δp affecting the regulation of ΔpH and generation of ΔΨ during fermentation of the mixture of glucose, glycerol and formate. Moreover, F_OF₁-lacking cells exhibited variation of metabolic end-products and demonstrated efficient energy transduction to save viability under fermentative energy-limited conditions.

Variable preferences towards low-nutrient conditions are typical for facultative oligotroph Thermus sp. Although, they are alkaliphilic bacteria, naturally hot springs are rich of different minerals and anions acidifying the environment. Understanding energy generation and pH stress responses in high thermal conditions are essential for unravelling energy conversion systems in thermophilic bacteria. Transmembrane pH differences and maintaining membrane potential are the first key energy conversions in living cells. Different complex structures are responsible for maintaining pH balance inside and outside of the cell, these structures include transporters, energy generation and modification. Therefore, measurements were done to determine specific growth rate, and aforementioned bioenergetic parameters under aerobic metabolism at slight acidic pH (6.0) in presence of sole carbon sources (glucose, fructose, lactose, starch) with concentrations of 2 g L^-1. In this study fluorescent probe (9-aminoacridine) for determination transmembrane pH difference, ion selective TPP⁺ (tetraphenylphosphonium ion) electrode for membrane potential determination, and spectrometry for specific growth rate were applied. Results shown that T. scotoductus maintain cellular pH between 6-7.5, showing the highest values in presence of glucose and starch, respectively, pH_in ~7.56 and ~7.61, showing ΔpH (difference between pH_in and pH_out) of ~1 and ~1,4, therefore these sugars play important role in ΔpH generation. On the other hand, reference sample with no sugar added has only negative ΔpH ~0.24, and membrane potential ~-50 mV, showing cell surviving strategy in energy limited condition for provoding biomass production. Surprisingly, negative delta pH was observed in presence of lactose (~1.05), which implies exergonic reaction processes to maintain pH differences and membrane potential, shown same pattern as in most conditions. Besides, again in presence of starch membrane potential was higher (~-64 mV), compared to other conditions, where the changes were not significant, respective to no sugar added sample (~-50 mV). Perhaps, acidic pH promotes starch utilization processes. Same promotion was shown in specific growth rate results at pH 6.0, where in presence of starch it was the highest, compared to no sugar added sample. To conclude, pH stress response of T. scotoductus K1 depends on environmental composition, particularly the types of carbon sources present which influence membrane potential and ΔpH fluctuations. These studies hold significance in describing and reporting the peculiarities of carbon source depended cell growth in high thermal conditions serving valuable insights for both basic research and biotechnologies.

Although there is evidence that in animals adapted to high altitude (HA), cellular oxygen utilization is optimized, the potential plasticity of metabolic pathways during postnatal development is unknown. Because, contrary to rats, mice are a model of successful HA adaptation, we used high resolution respirometry to measure mitochondrial oxygen consumption rates (OCR), mitochondrial protein concentration and citrate synthase activity in the cerebral cortex at postnatal days 7 (P7), P14 and P21, and at adulthood under sea level conditions (SL – Québec, Canada) and at HA (La Paz, Bolivia, 3600m). O2 consumption rates were measured under states of proton LEAK (OCR_LEAK) and oxidative phosphorylation with substrates of complex I (OCR_N), II (OCR_S) or I+II (OCR_NS). Oxidative capacity was also calculated as the difference OCR_NS – OCR_LEAK and represents the net O₂ capacity available for ATP synthesis. Adult mice and rats raised at HA exhibit higher oxidative capacity than SL animals, linked to a decrease OCR_LEAK in mice and an increase OCR_NS in rats. Throughout postnatal development, HA mice have a very high OCR_LEAK associated with a lower OCR_s and oxidative capacity at P7_. Conversely, rats raised at HA show higher OCR_LEAK at P7 only and increased OCR_NS, OCR_N and OCR_S at P7 and P14, leading to higher oxidative capacity than in SL animals. Furthermore, mitochondrial protein concentration and citrate synthase activity were not affected at P7 but were largely increased at P14 in HA rats compared with SL animals, suggesting that OCR might be supported by more efficient mitochondria at P7 and higher mitochondrial content at P14. Such differences during postnatal development and at adulthood observed in animals showing divergent responses to HA underlie the potential roles of theses responses in immature animals as a key element of HA adaptation.

Hydrogen sulfide (H₂S), as nitric oxide (NO), is a physiologically relevant, yet potentially toxic, endogenous gaseous signalling molecule of energy metabolism and other cellular pathways. The role of H₂S in cell physiology, while thoroughly investigated in mammals, awaits to be assessed in bacteria. Unlike Eukaryotes, bacteria can survive in H₂S-rich microenvironments thanks to a H₂S-insensitive respiratory oxidase cytochrome bd [1]. Some of them, but not the multidrug-resistant pathogen Pseudomonas aeruginosa [2], reportedly use endogenous H₂S to defend themselves against antibiotics and oxidative stress [3]. Targeting H₂S metabolism was therefore suggested to be a possible antibacterial strategy [4]. In bacteria H₂S is detoxified to sulfite and (thio)sulfate by a multienzymatic unit (located in mitochondria in Eukaryotes), which comprises persulfide dioxygenase (PDO), a non-heme Fe containing enzyme that metabolizes glutathione persulfide and O₂ to reduced glutathione and sulfite. Here, the PDO from Pseudomonas aeruginosa was structurally and functionally characterized by X-ray crystallography and high-resolution respirometry combined with NO amperometry and unexpectedly found to be potently and reversibly inhibited by NO, suggesting a novel crosstalk mechanism between H₂S and NO.

[1] M.R. Nastasi, L. Caruso, F. Giordano, M. Mellini, Cyanide insensitive oxidase confers hydrogen sulfide and nitric oxide tolerance to Pseudomonas aeruginosa aerobic respiration, Antioxidants (Basel), 13 (2024) 383

[2] L. Caruso, M. Mellini, O. Catalano Gonzaga, A. Astegno, E. Forte, A. Di Matteo, A. Giuffrè, P. Visca, F. Imperi, L. Leoni, G. Rampioni, Hydrogen sulfide production does not affect antibiotic resistance in Pseudomonas aeruginosa, Antimicrob Agents Chemother., 68 (2024) e0007524

[3] K. Shatalin, E. Shatalina, A. Mironov, E. Nudler, H₂S: a universal defense against antibiotics in bacteria, Science, 334 (2011) 986–990.

[4] K. Shatalin, A. Nuthanakanti, E. Nudler, Inhibitors of bacterial H₂S biogenesis targeting antibiotic resistance and tolerance. Science, 372 (2021) 1169-1175.

Modified tetrapyrroles are large cyclic macromolecules pivotal to a variety of biological processes, including electron transfer and microbial energy conservation solutions. Hemes b, a class of tetrapyrroles ubiquitous across all domains of life, play indispensable roles in these processes. For a long time, it has been assumed that heme biosynthesis exists as a sole conserved pathway in all organisms capable of synthesizing these compounds. However, additional pathways for heme biosynthesis have been unveiled since. Currently, there are three recognized heme biosynthesis pathways, each named after the key intermediate preceding the heme b formation.

Despite decades of research into heme b biosynthesis, no comprehensive large-scale evolutionary analyses of these pathways have been performed. Furthermore, the research was predominantly focused on heme biosynthesis and uptake within eukaryotes or well-studied prokaryotes, neglecting the less studied and newly discovered taxa. With the rapid technological developments in metagenomics and sequencing technologies over the past two decades, it became urgently necessary to conduct large-scale genomic analyses to obtain a more comprehensive representation of the organismal diversity derived from metagenomics. This project addresses this issue by employing methods of comparative genomics on metagenomic data to resolve the distribution, composition, and evolution of hemes biosynthesis pathways in prokaryotes. In this poster, we present an analysis of heme biosynthesis pathways across 35000 prokaryotic assemblies, offering insights into their evolution.

Mitochondrial function depends on direct interactions between respiratory proteins encoded by genes in two genomes, mitochondrial and nuclear, which evolve in very different ways. Serious incompatibilities between these genomes can have severe effects on development, fitness and viability. The effect of subtle mitonuclear mismatches has received less attention, especially when subject to mild physiological stress, such as high-protein diet, though these subtle mismatches are arguably more relevant to both evolutionary population dynamics and precision medicine. Here we show that subtle differences in mtDNA (1-9 SNPs) between Drosophila melanogaster lines that are isogenic in their nuclear background can cause substantial differences in lifespan, fertility and physical activity. These phenotypic differences were amplified by a high protein diet. Even though the genotypic differences between our fly lines affect respiratory-chain function alone, we only detected subtle variations in substrate use and complex I function using high-resolution O2k fluorespirometry in the thoraces and reproductive tissues of young and old flies of both sexes. These subtle variations underpinned marked differences at the level of metabolic flux and especially gene expression, with changes in core metabolism including Krebs cycle and glycolysis as well as purine and pyrimidine pathways. Differences in gene expression were especially marked in the reproductive tissues of both sexes, and in older flies. Surprisingly, the line with the most mitonuclear mismatches also had (slightly) the best complex I function in young adults, which corresponded to the longest lifespan, the greatest fertility and the most physical activity. This suggests that subtle differences in substrate preference can have concerted effects on metabolic plasticity, gene expression and epigenetic state. Better complex I function may enhance metabolic plasticity and life-history outcomes. These outcomes are not easily predicted on the basis of mitonuclear match alone, but a better understanding of respiratory flux will have important implications for both evolutionary biology and health.

Serpentinization in hydrothermal vents is central to some autotrophic theories for the origin of life because it generates compartments, reductants, catalysts and gradients. During the process of serpentinization, water circulates through hydrothermal systems in the crust where it oxidizes Fe (II) in ultramafic minerals to generate Fe (III) minerals and H₂. Molecular hydrogen can serve as a source of electrons for the reduction of CO₂ to organic compounds, with suitable catalysts present. Using catalysts during serpentinization H₂ reduces CO₂ to formate, acetate, pyruvate, and methane. These compounds represent the backbone of microbial carbon and energy metabolism in acetogens and methanogens, anaerobic chemolithoautotrophs that use the acetyl-CoA pathway of CO₂ fixation and that inhabit serpentinizing environments today. Serpentinization generates reduced carbon, nitrogen and probably reduced phosphorous compounds that were likely conducive to the origins process. It gives rise to microcompartments and proton gradients to support chemiosmotic ATP synthesis by the rotor-stator ATP synthase. This would help to explain why the principle of chemiosmotic energy harnessing is more conserved than the machinery to generate ion gradients via pumping coupled to exergonic chemical reactions, which in the case of acetogens and methanogens involve H₂-dependent CO₂reduction. Serpentinizing systems exist in terrestrial and deep ocean environments and were probably even more abundant on the early Earth. Serpentinization once occurred on Mars and is likely still occurring on Saturn’s icy moon Enceladus, providing a perspective on serpentinization as a potential source for life on other worlds.

The membrane-bound hydrogenase (Mbh) from Pyrococcus furiosus is an archaeal member of the Complex I superfamily [1-3]. Mbh catalyzes the reduction of protons to form H₂ gas, which is further functionally coupled to ion pumping (Na⁺/H⁺) across the membrane [2]. The [NiFe] cluster in the hydrophilic domain of Mbh is responsible for the evolution of H₂ gas, with both similarities and key differences compared to canonical soluble [NiFe] hydrogenases. In this work, we elucidate the molecular principles of H₂ production using quantum density functional theory and showcase, through large-scale molecular dynamics simulations, how H₂ catalysis transduces signals to the membrane arm through an intricate network of loops. We investigate all catalytic intermediates (Ni- SI_a, Ni-L, Ni-C, and Ni-R) of the [NiFe] active site, as well as the effect of different spin states [1], and demonstrate that the proton transfer reactions during catalysis are gated by the electric field effects. We observe that the protons required for H₂ evolution can be transferred via the glutamate (Glu21_L) pathway, whereas the histidine pathway (His75_L) becomes active during the second proton delivery step. These local proton-coupled electron transfer (PCET) reactions further induce functional conformational changes particularly in the highly conserved β1-β2 loop, which transduces the redox energy into ion-transport [3] across the membrane-arm via a conserved loop network. In summary, Mbh employs redox-triggered electric field effects to power the catalysis and ion-pumping, with general coupling principles that are similar to other members of the Complex I superfamily.

[1] A. Sirohiwal, A. P. Gamiz-Hernandez, V. R. I. Kaila, Mechanistic principles of hydrogen evolution in the membrane-bound hydrogenase, bioRxiv, 03.16.585322 (2024)
[2] M. E. Mühlbauer, A. P. Gamiz-Hernandez, V. R. I. Kaila, Functional Dynamics of an Ancient Membrane-Bound Hydrogenase, J Am Chem Soc, 143 (2021) 20873-20883.
[3] H. Yu, C. H. Wu, G. J. Schut, D. K. Haja, G. Zhao, J. W. Peters, M. W. W. Adams, H. Li, Structure of an Ancient Respiratory System, Cell, 173 (2018) 1636-1649.

Benzoyl-CoA reductases (BCRs) catalyse the reduction of benzoyl-CoA to 1,5-dienoyl-CoA, a key reaction in the anaerobic degradation of aromatic compounds, which occurs at a redox potential of E°’ = −620 mV. This is well below all known physiological electron donors [1]. Two classes of BCRs have been identified: While class I BCRs couple the endergonic reduction of benzoyl-CoA to ATP hydrolysis in facultative anaerobes, the structurally unrelated class II BCRs catalyse this reaction in obligate anaerobes via a proposed flavin-based electron bifurcation [2]. Class II BCRs have been enriched from Geobacter metallireducens and Desulfosarcina cetonica and consist of eight subunits with the composition of Bam[(BC)₂DEFGHI]₂ [3,4]. The subunits share similarities with aldehyde: ferredoxin oxidoreductases (BamB), electron-bifurcating heterodisulfide reductases (BamDE), and electron-bifurcating FeFe-hydrogenases (BamGHI). With 4 tungstopterins, 2 selenocysteins, 4 FADs, 2 FMNs and >50 FeS clusters, they are among the most complex metalloenzyme machineries known [3,4].

We used cryo-electron microscopy to gain insights into the largely unknown structure and function of this complex and obtained structures of the G. metallireducens and D. cetonica class II BCRs. The arrangement of the electron input and output modules suggests that class II BCRs achieve enzymatic dearomatisation by two successive electron confurcation/bifurcation processes: NADH and reduced ferredoxin act as donors for an electron confurcation at a flavin located in BamH. This is followed by an electron bifurcation at an FAD in BamE to benzoyl-CoA bound to BamB [1] and an unknown high potential acceptor. The latter is likely to be an electron-transferring flavoprotein as predicted by AlphaFold.

[1] Kung et al. (2010), JACS, 132(28), 9850–9856

[2] Kung et al. (2009), PNAS, 106(42), 17687–17692

[3] Huwiler, Löffler, Anselmann et al. (2019), PNAS, 116(6), 2259–2264

[4] Anselmann et al. (2019), Environ. Microbiol., 21(11),4241–4252

Potential differences for protein-mediated electron transfer through biopolymers or in bio-nano setups can amount to several 100 mV (respiratory complex I, nitrite reductase, light-induced processes), they lie far outside the range of linear response theory. We describe these situations by Pauli-master equations that are based on classical Marcus’ theory of electron transfer and Kirchhoff’s circuit laws. Furthermore, we take into account on-site blockade effects and a full non-linear response of the local potentials. We provide analytical and numerical current-potential curves and electron populations for multi-site model systems and biological electron transfer chains based on heme molecules or iron-sulfur clusters [1].

[1] M. Castellano, C. Kaspar, M. Thoss, T. Koslowski, Protein charge transfer far from equilibrium: a theoretical perspective, Phys. Chem. Chem. Phys., 25 (2023) 30887-30896.

Electron bifurcation is a fundamental energy coupling mechanism, widespread in anaerobes [1]. In acetogenic bacteria, electron-bifurcating [FeFe] hydrogenase HydABC complex links the H₂ oxidation with the generation of the reducing equivalents, ferredoxin (Fd) and NADH. Both the Fd and NADH, are reoxidized by reducing CO₂ to acetate in the Wood–Ljungdahl pathway, whereas Fd is also used for powering the primary respiratory enzyme to generate a chemiosmotic gradient for ATP synthesis. Recently, we revealed the structure and mechanism of the HydABC complexes [2]. However, the HydBC subcomplex is extremely modular and diverse across many anaerobes. The module attaches itself to various electron input subunits and couples itself reversibly to the reduction of Fd and NAD⁺. To explore its diversity, we determined cryo-EM structures of two other electron bifurcating complexes which harbour the HydBC-like modules. The structure of the electron-bifurcating transhydrogenase StnABC, which links the redox pools of Fd, NADH, and NADP⁺, reveals a modular amalgamation of different proteins. The three-subunit StnABC consists of the StnAB subcomplex, which is similar to the electron-bifurcating HydBC module, while StnC is a large protein possessing a NuoG-like domain and a GltD-like NADPH binding domain that strongly resembles NfnB of the NfnAB complex but cannot bifurcate electrons [3]. On the other hand, the architecture of the electron bifurcating formate dehydrogenase-hydrogenase complex (HytABCDE₁E₂) reveals a unique assembly. The complex forms a dimeric core consisting of a hydrogenase and formate dehydrogenase, which are flanked by the HydBC module to reversibly couple the oxidation of Fd and NADH for the production of formate from CO₂ or H₂ from protons. In sum, our work highlights how modular evolution in anaerobic metabolism produces novel activities critical for survival at the thermodynamic limit of life.

[1] V. Müller et. al. Electron bifurcation: a long-hidden energy-coupling mechanism, Annu. Rev. Microbiol., 72 (2018), pp. 331-353.

[2] A. Katsyv^†, A. Kumar^†, P. Saura^†, et. al. Molecular Basis of the Electron Bifurcation Mechanism in the [FeFe]-Hydrogenase Complex HydABC, JACS 2023 145 (10), 5696-5709.

[3] A. Kumar et. al. Molecular architecture and electron transfer pathway of the Stn family transhydrogenase. Nat Commun 14, 5484 (2023).

The yeast R. mucilaginosa is a highly resistant organism found in diverse extreme environments ranging from the ice in Antarctica to highly contaminated industrial wastewaters containing heavy metals. It was decided to analyze the possible mechanisms underlying this resiliency. We determined the structure of its highly branched mitochondrial respiratory chain and the ability of its endogenous carotenoids to deactivate reactive oxygen species (ROS). The branched mitochondrial respiratory chain (RC) in this yeast expresses all four orthodox complexes, plus different alternative redox enzymes including two type-2 NADH dehydrogenases (ND2), a glycerol-3-phosphate dehydrogenase, a cytochrome c-dependent lactate dehydrogenase and an alternative oxidase (AOX). Furthermore, R. mucilaginosa also expresses a cytoplasmic fumarate-reductase that seems to partition to mitochondria during the log-growth phase. Remarkably, respiratory activity decreases in the stationary phase. In regard to carotenoid production, R. mucilaginosa produces basal concentrations of three carotenoids (beta-carotene, thorularhodin and torulene) that increase when oxidative metabolism is enhanced by feeding cells with the respiratory substrate lactate. Oxidative metabolism increases ROS concentrations that are inactivated by carotenoids. Expression of each of these protection systems seems to be highly regulated and coordinated. Exceptional protection against stress resulted from the combination of these two systems, i.e., while respiratory chain branching prevented ROS overproduction, carotenoids inactivated the remaining free radicals.

Light-induced oxidative damage is known as photodegradation or photodecomposition which plays an important role in carbon cycling by modifying the bio-availability of energy stored in plant material for further decomposition by microbiota. Photodegradation does not depend on functional electron transfer systems of photosynthesis and respiration. The light dependence of electron transfer systems are measured via so-called photosynthesis-irradiance (PI) curves up to saturating light intensity. This study presents a novel approach to measure oxygen consumption-irradiance (OCI) curves under controlled light conditions in preparations of leaf samples.

Leaves were collected from a Robinia pseudoacacia tree in autumn and prepared for high-resolution respirometry with the Oroboros NextGen-O2k equipped with the PhotoBiology Module. In fresh green leaves cut into small pieces, PI curves showed a compensation point at a light intensity of 300 µmol·s^-1·m^-2. Green and yellow senescent leaves were homogenized, frozen at -20 °C, and measured at 20 °C [1].

Unexpectedly, oxygen consumption in the leaf homogenates increased with light intensity, saturating in the range of 1300 to 2000 µmol·s^-1·m^-2. Remarkably, the shape of the OCI curves mirrored PI curves of intact photosynthetic systems. Application of selective inhibitors targeting the electron transfer system of mitochondria provided evidence against their involvement in dark and light-induced oxygen consumption. Oxygen consumption, therefore, was assigned as photodegradation. Oxygen kinetics at 2000 µmol·s^-1·m^-2 were biphasic, with a linear increase above 8 µM O₂ and a hyperbolic phase with an “apparent K_m” (oxygen concentration c₅₀ at half-maximal flux) of ca. 0.9 µM, surprisingly close to the c₅₀ range of dark respiration through the cytochrome c oxidase and alternative oxidase pathway [2].

Future investigations will explore the effects of specific inhibitors of the chloroplast electron transfer system on photodegradation. Additionally, studies incorporating sample preparations preserving mitochondrial and chloroplast functional integrity in leaves at different life cycle stages will integrate our findings into the broader context of plant physiology and ecosystem carbon balance.

[1] Hardorp RA (2024) Oxygen consumption and production measured by high-resolution respirometry in leaf preparations from Robinia pseudoacacia. Bachelor-Thesis 47 pp. - https://www.mitofit.org/index.php/Hardorp_2024_Bachelor-Thesis

[2] Huete-Ortega M, Di Marcello M, Iglesias-Gonzalez J, Gnaiger E (2020) High-resolution respirometry for chloroplast and mitochondrial bioenergetics in Chlamydomonas reinhardtii - towards biotechnology exploitations. MitoFit Preprint Arch EA20.1. https://doi.org/10.26124/mitofit:ea20.algaeurope.0001

Almost all life on earth depends on photosynthesis, which is the basis of agricultural food and feed production. Regulation of photosynthesis is fundamental for efficiency and coping with changing environmental factors, preventing excess light energy from causing oxidative damage. Alternative electron pathways are crucial “safety valves” under stressful conditions, including changes in irradiation, nutrient availability and potentially high oxygen tensions. Algae use flavodiiron proteins (FDP) to protect photosystem I (PSI) by diverting excess electrons to reduce oxygen to water without forming reactive oxygen species (ROS). In Chlamydomonas reinhardtii, they are considered to protect PSI during a dark to light transition when the Calvin-Benson cycle is not active, or under fluctuating light. Oxygen tensions of the water column can vary considerably; especially dense algal populations exposed to high light and CO₂ concentrations encounter hyperoxia due to high photosynthetic rates. Here, using high precision respirometry, PSI absorption measurements (P700) and an FDP knockout (flvb), we further characterised FDP activity and its protective role. Near-infra red absorbance measurement of the PSI reaction centre (P700) of C. reinhardtii, indicated that hyperoxia led to P700 oxidation during a saturating pulse in wild-type, but not in an FDP-deficient mutant (flvb). We therefore hypothesized that FDP play a role under hyperoxia and saturating light intensities, protecting from excess Mehler reaction and ROS formation. While results supported our hypothesis, to our surprise we found that under constant sub-saturating light intensity that FDP is actually important in the protection of PSII.

Photosynthetic organisms drive the life cycle and carbon fixation on Earth. Inhabiting extremely diverse environments, they always have to cope with a friend that is simultaneously their enemy: the sunlight. Photon energy is necessary for photosynthesis, but it continuously damages the photosynthetic apparatus, primarily the first enzyme in oxygenic photosynthesis – Photosystem II (PSII) – in the process of photoinhibition. Upon photodamage, PSII becomes irreversibly inactivated, unable to do electron transfer, and requires costly repair involving protein translation. Two major types of photoinhibition are thought to exist – a donor-side mechanism, where the oxygen-evolving cluster absorbs UV light and becomes inactivated; and a charge recombination-driven, ¹O₂-producing pathway that damages the reaction centre of PSII.

Here we present an investigation of the efficiency of non-photochemical quenching – a regulatory process which decreases the lifetime of chlorophyll excited states – in PSII photoprotection. We show an apparent inefficiency in its function, in line with early work [1,2]. We further examine the effect of PSII bioenergetics on the rate of photodamage, using a novel approach where PSII rate is normalised among a number of different photosynthetic microbes. This allows comparisons of photoinhibition rates in species where non-canonical chlorophyll molecules [3] are employed in the antenna and the PSII reaction centre. The results are consistent with a charge separation-independent mechanism of PSII photoinhibition.

[1] Santabarbara, S., Cazzalini, I., Barbato, R., Tarantino, D., Zucchelli, G., Garlaschi, F. and Jennings, R., 2001. Is non photochemical quenching protective?. Science Access, 3(1).

[2] Santabarbara, S., Neverov, K.V., Garlaschi, F.M., Zucchelli, G. and Jennings, R.C., 2001. Involvement of uncoupled antenna chlorophylls in photoinhibition in thylakoids. FEBS letters, 491(1-2), pp.109-113.

[3] Viola, S., Roseby, W., Santabarbara, S., Nürnberg, D., Assunção, R., Dau, H., Sellés, J., Boussac, A., Fantuzzi, A. and Rutherford, A.W., 2022. Impact of energy limitations on function and resilience in long-wavelength Photosystem II. Elife, 11, p.e79890.

The superoxide anion is classified as reactive oxygen species (ROS) which delineate a group of oxygen containing, chemically reactive molecules. It is produced either enzymatically on purpose or by adventitious single electron transfer reactions to molecular oxygen, particularly by enzymes of the respiratory chain. While ROS are known to have pivotal cellular functions at low concentrations in signaling pathways, excessive levels can induce cellular damage and lead to oxidative stress implicated in various diseases. In 2018, we contributed to the description of the membrane-embedded protein cytochrome b561 (CybB) from Escherichia coli, being only the third enzyme known to scavenge superoxide. CybB acts as a superoxide:quinone oxidoreductase channeling electrons from periplasmic superoxide via two b-type hemes to quinones in the membrane. Remarkably, CybB can also function in the “reverse” direction and serve as a quinol consuming superoxide producer, thus exhibiting dual functionality. Given the molecular structure, both reactions are anticipated to be electrogenic due to the transport of electrons across the membrane. We consider the electrogenicity of the reaction as a central aspect for a recently found growth phenotype in ΔcybB knockout cells. To experimentally verify electrogenic enzyme activity aimed to monitor membrane potential spectrophotometrically using potentiometric dyes. The stoichiometry of the reaction mechanism demands binding of two superoxide molecules (equivalent to two electrons) and uptake of two protons per quinol reduced. So far, detailed understanding of the reaction sequence was limited by to the very rapid kinetics and hard-to-control superoxide concentrations. Pulse radiolysis allows to precisely generate defined amounts of superoxide within nanoseconds, enabling us to elucidate kinetic rates and identify critical residues for the reaction.

The nicotinamide nucleotide transhydrogenase is present in the mitochondrial inner membrane and in the cytoplasmic membranes of many bacteria. The enzyme catalyzes the reversible hydride transfer between NAD(H) and NADP(H) and couples this reaction to the proton motive force to promote the generation of NADPH. The enzyme consists of three domains: domain I binds NAD(H); domain II contains multiple transmembrane helices and a proton channel; domain III binds NADP(H). Large conformational changes of the enzyme isolated from ovine mitochondria were previously observed in the presence of NADP⁺ or NADPH, but none of the conformations are compatible with hydride transfer from NADH to NADP⁺. In this work, the structure of the transhydrogenase from E. coli has been determined by cryo-electron microscopy, capturing multiple conformations in the presence of ligands. Most important is a conformation observed in the presence of a mixture of NADPH and NADP⁺ in which the binding sites for NAD(H) (domain I) and NADP(H) (domain III) are adjacent and compatible with direct hydride transfer.

Efficiency can be defined as the amount of useful energy delivered by a system relative to the total energy supplied. In metabolism, this concept is exemplified by the amount of ATP produced per substrate molecule. Aerobic processes are crucial in biotechnology for generating anabolic products such as biomass and proteins. These processes are intrinsically linked to ATP yield on substrate. Not only are yields on substrates essential, but yields on oxygen are also critical; large-scale aerobic processes often face the challenge of oxygen limitation, which impacts cellular metabolism and negatively affects production.

Yields are partially governed by the efficiency of electron transport chains, specifically P/O ratios (ATP molecules synthesized per electron pair used to reduce ½ O₂). On the one hand, the maximal theoretical P/O ratio in yeasts ranges from 1.4 to 2.3, whereas for mammalian cells it is accepted to be 2.7. On the other hand, yeasts and other microorganisms have faster growth rates and higher yields, and require less nutrient supplementation than mammalian cells, making them great hosts for large-scale production.

My newly established research group aims to address such energetic paradoxes in industrially relevant cells, focusing on both basic and applied science. Central to our approach is the quantitative analysis of cellular physiology, which is inherently growth-dependent. For this, we make use of bioreactor cultivation. Engineering energetic efficiency involves editing the mitochondria in eukaryotes. Although most proteins in the organelle are encoded by the nuclear genome, some are still retained in the mitochondrial genome. We are implementing biolistic transformation approaches for mitochondrial DNA editing, and respirometry systems for detailed mitochondrial characterization. We also make use of analytic tools together with modelling approaches to quantify cellular fluxes and control, and proteome efficiency in metabolic routes.

Current projects include exploring alternative feedstocks, investigating yeast diversity and enhancing biomass yields. A particular focus is on the composition of the electron transport chain in relation to cellular yields in yeasts [1]. In addition, we are initiating new projects on F₁F_o‑ATP synthase engineering and also on the efficiency of mammalian cells in bioreactors for bioproduction.

[1] Warmerdam, M. Specific growth rates and growth stoichiometries of Saccharomycotina yeasts on ethanol as sole carbon and energy substrate (2024) Manuscript in preparation.

Ethanol is produced at industrial scale from non-agricultural feedstocks by gas fermentation, while research on other low-emission processes for ethanol production is accelerating. In view of its degree of reduction, water solubility and relatively low toxicity, ethanol is an interesting candidate to replace sugars in aerobic, zero-emission processes for yeast-based production of whole-cell protein and low-molecular-weight compounds. Currently, little information is available on specific growth rates, biomass yields and biomass composition of yeast species during growth on synthetic medium with ethanol as sole carbon source. In this study, strains of 52 Saccharomycotina yeasts were screened for their growth characteristics on ethanol. After first screening in microtiter plates, 21 fast-growing strains that were further analysed in aerobic shake-flask cultures showed specific growth rates of 0.12-0.46 h^-1. Five fast-growing strains were further studied in aerobic, ethanol-limited chemostats (dilution rate 0.10 h^-1). Strains of the industrial yeasts Saccharomyces cerevisiae and Kluyveromyces lactis, whose genomes lack genes for a proton-coupled Complex-I NADH dehydrogenase, both showed biomass yields of 0.6 g biomass (g ethanol)^-1. Of three yeasts whose genome does contain Complex-I genes, Phaffomyces thermotolerans, showed the same biomass yield as S. cerevisiae, while Ogataea parapolymorpha and Cyberlindnera jadinii showed biomass yields of 0.67 ± 0.01 and 0.73 ± 0.00 g g^-1, respectively. The biomass yield of C. jadinii, which also showed the highest protein content of the 5 yeasts tested in chemostats, corresponded to 88% of the theoretical biomass yield in a scenario where growth is limited by assimilation rather than by energy metabolism.

Respiratory complex I (NADH:ubiquinone oxidoreductase) is the starting point for biochemical energy conversion in bacteria and mitochondria and maintains approximately 40% of the proton gradient driving ATP synthesis by oxidising NADH and reducing ubiquinone.

Deficiency of complex I causes a wide range of diseases such as cardiomyopathy and lactic acidosis[1] and changes in complex I function are implicated in the proliferation of cancer cells[2]. A better understanding of its structure, function and assembly are essential to develop treatments for these conditions and due to its structural similarity, yeast can serve as a model organism for mammalian complex I.

We show here four distinct conformational states of the aerobic yeast Yarrowia lipolytica under turnover conditions, including closed, open and intermediate states at resolutions up to 2.3 Å. Similar to E. coli complex I, we found closed state only under turnover conditions while in the apo-state, the majority of the protein adopts the open-ready conformation that was previously observed only in E. coli[3]. Moreover, while mammalian complex I shows a strong deactive conformational state that is thought to exist primarily to protect from ischemia/reperfusion injury and reduce damaging ROS generation, we did not find this state in Yarrowia lipolytica.

[1] Swalwell, H., Kirby, D., Blakely, E. et al. Respiratory chain complex I deficiency caused by mitochondrial DNA mutations. Eur J Hum Genet vol 19 (2011), 769–775

[2] Sollazzo, M., De Luise, M., Lemma, S., Bressi, L., Iorio, M., Miglietta, S., Milioni, S., Kurelac, I., Iommarini, L., Gasparre, G. and Porcelli, A.M., Respiratory Complex I dysfunction in cancer: from a maze of cellular adaptive responses to potential therapeutic strategies. FEBS J vol 289 (2022), 8003-8019

[3] Kravchuk, V., Petrova, O., Kampjut, D., Wojclechowska-Bason, A., Breese, Z., Sazanov, L., A universal coupling mechanism of respiratory Complex I, Nature, vol 609 (2022), 808-837

Skeletal muscle mitochondria are sensitive to intracellular signals to increase ATP production, with calcium (Ca++) being a key signaling molecule. A rise in cytosolic Ca++ results in mitochondrial uptake, with physiological Ca++ levels activating mitochondrial ATP production and supraphysiological levels leading mitochondrial permeability transition pore (MPTP) opening and loss of function. While these functional changes occur in seconds, it remains unknown if reorganization of mitochondrial complexes, particularly ATP synthase, can be acutely induced, and how intracellular signals affect this reorganization. PURPOSE: The goal of this study was to determine if activating and/or overload Ca⁺⁺ levels 1) induce changes in the organization of the mitochondrial complexes, specifically, the organization of CV into monomers (CV₁) and dimers (CV₂), 2) and if Pi plays a role in the regulation of CV₁ and CV₂ formation. METHODS: Mitochondria were isolated from skeletal muscle of Sprague-Dawley rats, then incubated in respiration media with glutamate + malate (sample aspirated), acutely subjected to activating Ca++ or overload Ca++ (samples aspirated), and then 10 mM Pi was added (samples aspirated). Aspirated mitochondria suspension samples were pelleted, solubilized (digitonin), and separated on a BN-PAGE gel to allow for the assessment of CV₁ & CV₂, analyzed via densitometry. RESULTS: Acute exposure of mitochondria to Ca++ resulted in a decrease in CV₁ and a concomitant increase in CV₂. Ca++ overload resulted in a larger decrease in CV₁ compared to Ca++ activation (CV₁ %76.34+5.4 vs 58.4±3.3), and a larger increase in CV₂ abundance (CV₂% 22.7+2.5 vs 35.3±3). CV₁abundance decreased further when Pi was added after Ca++ overload; however, CV1 abundance increased when Pi was added after Ca++ activation. Cyclosporin A, an MPTP blocker, did not prevent the decrease in CV₁ percentage during Ca++ overload, with or without the presence of Pi (CV₁% 55.6±5 vs 47.6±7, without Pi, p=0.069; 54.6±5 vs 42.7±6.6, with Pi, p=0.058).

CONCLUSION: Mitochondrial complex associations can be acutely altered by intracellular signals. Calcium activation and overload both appear to induce the CV to dimerize, and Pi plays a regulatory role in this dimerization. This modifiable, dynamic complex reorganization may play a key role in the mechanisms involved in MPTP opening.

Cells depend on a continuous and abundant supply of ATP, the universal energy currency that powers nearly all cellular reactions. In mitochondria, ATP is produced by a series of redox reactions, whereby protons are pumped across the inner mitochondrial membrane (IMM) to create an electrochemical gradient. The ATP synthase harnesses the energy of the proton gradient to generate ATP from ADP and inorganic phosphate. We determined the structure of ATP synthase within mitochondria of the unicellular green alga Polytomella at a local resolution of 5.6 Å using electron cryo-tomography and subtomogram averaging of thin lamellae cut out of flash-frozen cells. Our map reveals an additional, previously unknown density of the algae-specific subunit ASA3. 3D classification revealed six different rotary positions of the central stalk, subclassified into 21 substates of the F₁ head enabling us to describe the rotary movement of the enzyme under native working conditions. Analysing the higher order arrangement reveals that the mitochondrial ATP synthase forms helical arrays with multiple adjacent rows defining the cristae ridges. Row formation and stabilisation is achieved by a hook-shaped cleft formed by two Polytomella-specific subunits that extend towards the adjacent dimer, helping to hold the dimers in place while still allowing flexibility. Our study demonstrates the power of in situ structural biology that preserves interactions which may otherwise be lost by protein isolation and purification. It provides an essential basis for analysing in situ structures not only of bioenergetic complexes, but also of protein assemblies in the context of their native membrane.

Archaea from the Thermoproteota group, such as Sulfolobus acidocaldarius, are known to populate some of the most hostile environments on Earth, characterized by extreme temperatures and highly acidic ambient pH. These organisms utilize an unusual set of respiratory complexes, with surprising redundancy and unorthodox subunit assemblies. Thus far, three alternate terminal oxidases (SoxB, SoxM, and DoxB) and three cytochrome-ISP complexes (SoxCL2, SoxNL, and SoxGF) have been identified[1,2]. Herein, using a native source, we performed an extraction and partial purification of several respiratory enzymes of S. acidocaldarius: the NDH homolog, succinate dehydrogenase, and some of the terminal oxidase complexes. To obtain structural data on these enzymes we combine cryo-EM with novel computational tools: AlphaFold2[3] and AlphaFill[4]. Moreover, we designed a simple setup for functional studies on the terminal oxidase complexes, harnessing a heterologously produced sulfocyanin and the isolated native caldariellaquinone-6. We hope that our contribution will help to fill existing gaps in the knowledge of long-neglected archaeal respiratory enzymes.

[1] G. Schäfer, M. Engelhard, V. Müller, Bioenergetics of the Archaea, Microbiology and Molecular Biology Reviews 63 (1999) 570–620. https://doi.org/10.1128/mmbr.63.3.570-620.1999.

[2] L.F. Bischof, M.F. Haurat, L. Hoffmann, A. Albersmeier, J. Wolf, A. Neu, T.K. Pham, S.P. Albaum, T. Jakobi, S. Schouten, M. Neumann-Schaal, P.C. Wright, J. Kalinowski, B. Siebers, S.V. Albers, Early response of Sulfolobus acidocaldarius to nutrient limitation, Frontiers in Microbiology 10 (2019) 1–17. https://doi.org/10.3389/fmicb.2018.03201.

[3] J. Jumper, R. Evans, A. Pritzel, T. Green, M. Figurnov, O. Ronneberger, K. Tunyasuvunakool, R. Bates, A. Žídek, A. Potapenko, A. Bridgland, C. Meyer, S.A.A. Kohl, A.J. Ballard, A. Cowie, B. Romera-Paredes, S. Nikolov, R. Jain, J. Adler, T. Back, S. Petersen, D. Reiman, E. Clancy, M. Zielinski, M. Steinegger, M. Pacholska, T. Berghammer, S. Bodenstein, D. Silver, O. Vinyals, A.W. Senior, K. Kavukcuoglu, P. Kohli, D. Hassabis, Highly accurate protein structure prediction with AlphaFold, Nature 596 (2021) 583–589. https://doi.org/10.1038/s41586-021-03819-2.

[4] M.L. Hekkelman, I. De Vries, R.P. Joosten, A. Perrakis, AlphaFill: enriching AlphaFold models with ligands and cofactors, Nat Methods 20 (2023) 205–213. https://doi.org/10.1038/s41592-022-01685-y.

In most eukaryotic cells, several mitochondrial enzymes have been proposed as a hot point for ROS production, particularly the complexes of the electron transport chain. Recently, stable associations between respiratory complexes I, III₂, and IV have been described and named respirasome (Wittig et al., 2006); however, its precise participation in mitochondrial bioenergetics and ROS production is poorly understood. We have isolated the respirasome from Ustilago maydis and determined that it has a higher NADH:DBQ oxidoreductase activity coupled to a lower ROS production than the free complex I (Reyes-Galindo et al., 2019). To verify the role of respiratory complexes in ROS production, the respirasome, and free-complex I were isolated and incubated in the presence of seven of the most toxicologically relevant heavy metals to generate oxidative stress. Toxicological studies suggest that mitochondrial intoxication by heavy metals increases ROS production, reducing ATP synthesis and cell viability; however, the inhibition of respiratory complexes activities by heavy metals is still unknown. We showed a putative deactivation of the respirasomal-complex I by seven of the most toxicologically relevant heavy metals, without increasing the ROS production. In sharp contrast, the free complex I was more resistant to heavy metals but was 30 times more ROS-producing (de Lira-Sánchez et al., 2023). These results underly the preventive role of the respirasome in mitochondrial electron leak and ROS production and recall its disassembled in some pathologies which involve mitochondrial damage and oxidative stress.

This work was supported by Dirección General de Asuntos del Personal Académico (DGAPA, IN201923) from Universidad Nacional Autónoma de México (UNAM), and UC MEXUS-CONAHCYT collaborative grant (CN-20-327). de Lira-Sánchez is a Ph.D. student from Programa de Doctorado en Ciencias Biomédicas, UNAM, and received a fellowship (666472) from CONAHCyT.

The sodium-pumping NADH:quinone oxidoreductase (Na⁺-NQR) is six-subunit (NqrA, B, C, D, E, and F) respiratory complex of Vibrio cholerae. It oxidizes NADH and transfers the electrons to ubiquinone in a reaction analogous to mitochondrial complex I. However, the architecture of Na⁺-NQR is fundamentally different from complex I [1]. The energy derived from NADH oxidation it utilized by Na⁺-NQR to drive sodium translocation generating a sodium motive force that is required for motility or substrate uptake of the bacterium. We have determined a series of cryo-EM and X-ray structures of the Na⁺-NQR, which represent snapshots of the catalytic cycle [2]. We show that ion-pumping in Na⁺-NQR is driven by large conformational changes coupling electron transfer to ion translocation. Electrons are transferred by unique set of flavin and FeS cofactors that shuttle the electrons from NADH twice across the membrane to quinone. The redox state of a unique intramembranous [2Fe-2S] cluster orchestrates large conformational movements of different subunits in the complex. The structural and biochemical data put forward that large switching movements control the binding and release of the coupling ion Na⁺ visible in the structures.

[1] J.L. Hau, S. Kaltwasser, V. Muras, M.S. Casutt, G. Vohl, B. Claußen, W. Steffen, A. Leitner, E. Bill, G.E. Cutsail, S. DeBeer, J. Vonck, J. Steuber, and G. Fritz. Conformational coupling of redox-driven Na⁺-translocation in Vibrio cholerae NADH:quinone oxidoreductase, Nat Struct Mol Biol 30 (2023) 1686–1694. https://doi.org/10.1038/s41594-023-01099-0.
[2] J. Steuber, G. Vohl, M.S. Casutt, T. Vorburger, K. Diederichs, and G. Fritz. Structure of the V. cholerae Na⁺-pumping NADH:quinone oxidoreductase. Nature 516 (2014) 62–67. https://doi.org/10.1038/nature14003

NADH:ubiquinone oxidoreductase, respiratory complex I, couples the electron transfer from NADH to ubiquinone (Q) with the translocation of protons across the membrane. Escherichia coli complex I consists of 13 subunits called NuoA to NuoN. Electrons are transferred from NADH via FMN through a series of iron-sulfur (FeS) clusters to Q that is reduced and protonated in a unique binding chamber. This chamber comprises the hydrophobic subunits NuoA and NuoH and the hydrophilic subunits NuoB and NuoCD. A conserved Tyr residue and a conserved His residue have been proposed to be involved in Q binding and protonation. However, mutagenesis of these residues in Yarrowia lipolytica and E. coli led to inconclusive results [1,2]. Our data with the E. coli complex I support the findings presented for the Yarrowia enzyme [1]. In addition, titration with the specific inhibitor PiericidinA are in agreement with structural data [3] pointing to an involvement of the conserved His residue in inhibitor binding. As none of our E. coli mutants fully lost electron transfer activity, we conclude that the Tyr/His orients the Q in the binding pocket but are not involved in its protonation.

[1] L. Grgic, K. Zwicker, N. Kashani-Poor, S. Kerscher, U. Brandt, Functional significance of conserved histidines and arginines in the 49-kDa subunit of mitochondrial complex I, J. Biol. Chem. 279 (2004) 21193–21199. https://doi.org/10.1074/jbc.M313180200.

[2] H.R. Bridges, J.G. Fedor, J.N. Blaza, A. Di Luca, A. Jussupow, O.D. Jarman, J.J. Wright, A.N.A. Agip, A.P. Gamiz-Hernandez, M.M. Roessler, V.R.I. Kaila, J. Hirst, Structure of inhibitor-bound mammalian complex I, Nat. Commun. 11 (2020) 1–11. https://doi.org/10.1038/s41467-020-18950-3.

[3] P.K. Sinha, N. Castro-Guerrero, G. Patki, M. Sato, J. Torres-Bacete, S. Sinha, H. Miyoshi, A. Matsuno-Yagi, T. Yagi, Conserved amino acid residues of the NuoD segment important for structure and function of Escherichia coli NDH-1 (complex I), Biochemistry. 54 (2015) 753–764. https://doi.org/10.1021/bi501403t.

The cytochrome bd oxidases are triheme copper-free terminal reductases in bacterial respiratory chains that couple the oxidation of ubiquinol with the reduction of oxygen to water [1]. The production of bd oxidases is induced under oxygen limited growth conditions and they can degrade reactive oxygen or nitrogen species. Expression of bd oxidase increases virulence for example in Mycobacterium tuberculosis [2,3].Therefore, bd oxidases are considered as potential drug targets and special interest lies on their structure-function relationship [3]. Escherichia coli contains two cytochrome bd oxidases (bd-I and bd-II) that have a similar structure. However, it is not clear whether they also function in a similar way [1,4]. The bd oxidase’s proton pathways start at a hydrophilic cavity at the cytosolic side and lead to heme d in the membrane. Site-directed mutagenesis showed the importance of D105^CydB and D58^CydB (bd-I) and E58^AppB (bd-II) for the pathways [1,4]. The particular contribution to the pathways were elucidated. Several possible electron transfer routes between the heme groups are under debate. Here, strong interest lies on the heme b environment which features a highly conserved tryptophan residue proposed to be essential for electron transfer [1,5]. Point mutations at this position revealed that the Trp residue is indeed important for intramolecular electron transfer.

[1] A. Theßeling, T. Rasmussen, S. Burschel, D. Wohlwend, J. Kägi, T. Friedrich, Homologous bd oxidases share the same architecture but differ in mechanism, Nat. Commun., 10 (2019) 1-7.

[2] V. B. Borisov, E. Forte, Bioenergetics and Reactive Nitrogen Species in Bacteria, Int. J. Mol. Sci., 23 (2022) 7321.

[3] D. Bald, C. Villellas, P. Lu, A. Koul et al., Targeting energy metabolism in Mycobacterium tuberculosis, a new paradigm in antimycobacterial drug discovery, MBio, 8 (2017) 1-11.

[4] A. Grauel, J. Kägi, T. Rasmussen et al., Structure of Escherichia coli cytochrome bd-II type oxidase with bound aurachin D. Nat. Commun., 12 (2021) 1-11.

[5] W. Wang, Y. Gao, Y. Tang, X. Zhou et al., Cryo-EM structure of mycobacterial cytochrome bd reveals two oxygen access channels. Nat. Commun., 12 (2021) 1-8.

Mycobacterium smegmatis is a bacterial species closely related to Mycobacterium tuberculosis. Because of that it is widely used as a model organism to study for Mycobacteriacae. Throughout the recent years, mycobacterial electron transport chain (ETC) emerged as a potent target for antituberculosis drugs, especially its terminal enzymes, called terminal oxidases. Mycobacteria utilize two branches of the ETC: a bcc-aa₃supercomplex branch and a bd oxidase branch. While the first one plays a main role as the last enzyme catalyzing reduction of oxygen to water, the latter is crucial for the bacteria’s survival in conditions like very low oxygen level, high reactive oxygen species concentration, antibiotic stress etc. M. smegmatis, possesses two different cytochrome bd oxidases: cyt. bd-I and cyt. bd-II oxidase. The work presented during this conference shows that they can work with a different types of electron donors (quinols). Moreover, cyt. bd-II seems to be less conserved when it comes to the type of quinol it can work with which may suggest its yet unknown function in overtaking the role of cyt. bd-I during its inactivity. At the same time cyt. bd-II is more susceptible to already existing inhibitors of bacterial terminal oxidases like Aurachin D and to understand its nature better, we are also resolving its Cryo-EM structure. At 2.8 Å resolution, it is possible to see menaquinol molecules bound to the non-catalytic subunit AppB. Cyt. bd-II is very distinct from its paralogous cyt. bd-I in M. smegmatis, belonging to a different evolutionary group (qOR2) [1] and its function and conditions under it expresses are still not fully understood.

Proton-translocating respiratory supercomplexes provide the basis of safe and efficient energy conversion [1]. In cytochrome (cyt) bcc-aa₃ supercomplex, a di-haem cyt c bridges the electron transfer between cyt bccomplex (complex III) and cyt c oxidase (complex IV), which are drivers of the proton motive force that fuels ATP generation via respiration. In the 2.8 Å resolution cryo-EM structure of the obligate complex III₂-IV₂ (cyt bcc-aa₃) supercomplex of the actinobacterium Corynebacterium glutamicum [1], we elucidated the catalytic position for menaquinol oxidation and discovered two associated proton channels, a lycopene molecule, an acetylated phosphatidylinositol dimannoside (AcPIM₂), a lipomannan (Cg LM-A) fragment, a novel Q_c site occupied by menaquinone, and also identified the position of a dioxygen molecule in the oxygen tunnel of the oxidase. The data shed light on novel structural features and principles of catalysis in an obligate respiratory supercomplex. With the improvement of cryo-EM sample preparation, a new structure was obtained at high resolution enabling detailed analysis of lipid species, of the exact geometry of the catalytic centres, elucidating the adaptations of the supercomplex that spans from low potential menaquinol catalysis to oxygen reduction [2].

[1] W.-C. Kao et al., Structural basis for safe and efficient energy conversion in a respiratory supercomplex, Nat. Commun. 13, 545 (2022)

[2] W.-C. Kao and C. Hunte, Quinone binding sites of cyt bc complexes analysed by x-ray crystallography and cryogenic electron microscopy. Biochem. Soc. Trans. 50, 877-893 (2022)

Cyanobacteria are oxygenic photosynthetic bacteria that play a crucial role in the Earth's carbon cycle. The NDH complex, a key enzyme complex involved in photosynthesis, has been well-studied in cyanobacteria. It plays an important role in cyclic electron transfer (CET), during which electrons are cycled within PSI through ferredoxin and plastoquinone to generate proton gradient without NADPH production. Balance between NADPH and ATP is essential for the Calvin-Besnon cycle and varies under physiological conditions. NDH belongs to complex I family, however, the mechanism of it's action remains controversial.
Here we used cryo-electron microscopy (cryo-EM) to study the structure of the NDH complex from Thermosynechococcus elongatus. Our cryo-EM study revealed novel conformations of the NDH complex. The results suggest a dynamic behavior of the complex, which may play a role in regulating the photosynthetic process. These results have important implications for understanding of coupling mechanism of NDH and photosynthesis in general.

Cytochrome c oxidase (CcO) is a terminal membrane enzyme in the aerobic respiratory chain. CcO pumps protons against the membrane proton gradient and reduces oxygen to water using the energy of the electrons donated by cytochrome c [1]. It has been proposed that reduction of heme a in the catalytic cycle of CcO from Bos taurus leads to dissociation of the ion pair formed by an essential arginine and the heme a3 D-propionate, creating a strong field leading protons to a transient proton loading site (PLS) [2]. We mutated the highly conserved amino acid R473 (analogous to R438 in Bos Taurus), which forms salt bridges to the D-propionates of heme a and heme a3 in Paracoccus denitrificans [3], to a cysteine (R473C).

We investigated the effects of the R473C mutation on the electrostatic environment at the binuclear center. We monitored the changes induced by this mutation through electronic spectroscopy and vibrational spectroelectrochemistry [4]. The R473C mutant shows a decreased catalytic activity, blue-shifted Soret bands in both the oxidized and reduced states and minimal response to electrochemically-induced redox changes.

[1] M. Wikström, K. Krab, V. Sharma, Oxygen Activation and Energy Conservation by Cytochrome c Oxidase, Chem. Rev., 118 (2018) 2469-2490

[2] P. Saura, D. Riepl, D. M. Frey, M. Wikström, and V. R. I. Kaila, Electric fields control water-gated proton transfer in cytochrome c oxidase, Proc. Natl. Acad. Sci., 119 (2022) 1-9.

[3] J. Koepke, E. Olkhova, H. Angerer, H. Müller, G. Peng, H. Michel, High resolution crystal structure of Paracoccus denitrificans cytochrome c oxidase: New insights into the active site and the proton transfer pathways, BBA Bioener., 1787 (2009) 635-645.

[4] F. Baserga, J. Dragelj, J. Kozuch, H. Mohrmann, E. W. Knapp, S. T. Stripp and J. Heberle, Quantification of Local Electric Field Changes at the Active Site of Cytochrome c Oxidase by Fourier Transform Infrared Spectroelectrochemical Titrations, Front. Chem., 9 (2021) 2296-2646.

Oxidative phosphorylation is an indispensable pathway employed by the non-fermentative mycobacteria to sustain their energy requirements. The pathway oxidises electron carriers to establish an electrochemical gradient which is then harnessed to produce ATP and water. The formation of water is catalysed by the reduction of oxygen within the terminal oxidases: cytochrome cyt-bcc:aa₃ and cytochrome cyt-bd. The energetically favourable cyt-bcc:aa₃ orchestrates multiple key events and remains relevant and active even under hypoxia despite possessing lower affinity towards oxygen compared to cyt-bd, the substitute oxidase. The cyt-bcc:aa₃ oxidase assumes a unique supercomplex arrangement comprising of cyt-bcc (analogous to Complex III in mitochondria), cyt-aa₃ (analogous to Complex IV) and a superoxide dismutase. Here, we present atomic features of the first complete M. tuberculosis cyt-bcc:aa₃ supercomplex derived from a cryo-EM campaign which revealed electron-, oxygen and proton pathways. The findings from the study highlight mechanistic variations specific to M. tuberculosis that subsequently served as a foundation for structure-based drug discovery efforts.

[1] V. Mathiyazakan, C.F. Wong, A. Harikishore, K. Pethe, G. Grüber, Cryo-Electron Microscopy Structure of the Mycobacterium tuberculosis Cytochrome bcc:aa₃ Supercomplex and a Novel Inhibitor Targeting Subunit Cytochrome cI. Antimicrobial Agents and Chemotherapy, 67 (2023). doi:10.1128/aac.01531-22.

The mammalian oxidative phosphorylation (OXPHOS) system in the inner mitochondrial membrane comprises respiratory chain complexes I-IV (CI-CIV) and ATP synthase. Together, these protein complexes harvest metabolic energy to generate ATP, the cellular energy currency. The inner mitochondrial membrane is abundant in OXPHOS complexes and CI, CIII₂ and CIV exhibit specific protein-protein interactions to form stable supercomplex assemblies, exemplified by the respirasome (CI-CIII₂-CIV). Supercomplexes are conserved in evolution, and well documented by biochemical and structural methods. However, their physiological roles are much debated, despite substantial literature suggesting their importance in facilitating catalysis and regulating turnover in response to metabolic demand. To investigate the in vivo role of respirasomes, we deleted a short conserved, charged loop in the UQCRC1 subunit of CIII₂ that contacts CI. Different tissues in the resulting homozygous knock-in mice show profoundly decreased levels of respirasomes on blue native polyacrylamide gel electrophoresis (BN-PAGE) and complexome profiling proteomics analyses of different tissues, although the individual complexes appear unaffected. In vivo spatial organization of respirasomes was altered in knock-in mice as shown by cross-linking experiments on intact mitochondria. Surprisingly, the mutant mice appear healthy, with maintained respiratory chain capacity and exercise tolerance. Our results suggest that respirasomes have no major role in regulation of OXPHOS capacity in vivo.

Impaired mitochondria in cardiovascular diseases (CVDs) lead to enhanced oxidative stress, diminished ATP production, and impaired autophagic mechanisms. It is demonstrated that dietary habits play a role in CVD. A high salt diet greatly accelerates hypertension and stroke through complex I dysfunction in stroke-prone spontaneously hypertensive rats (SHR-SP) [1]. We try to evaluate the kinetic activity of the respiratory complexes of rat heart mitochondria of SHR-SP and SHR stroke-resistant (SHR-SR) strains fed with normosodic (ND) or Japanese Diet (JD). Freeze-thawed rat heart mitochondria were investigated by metabolic control analysis to detect the control coefficient (Ci) on the flux control exerted by complex IV (CIV) over aerobic NADH oxidation determined using KCN to progressively inhibit NADH-O₂ and TMPD/ASC-O₂ oxidoreductase activity [2]. Under ND conditions, NADH-driven respiratory rate of SHR-SR or SHR-SP showed a Ci of 0.97 or 0.89, respectively. The data corroborated the linear threshold plots highlighting that high control is exerted by the terminal enzyme of respiratory chain in SHR heart mitochondria. Otherwise, SHR-SR and SHR-SP treated with JD underwent different control of the aerobic oxidation of NADH. SHR-SR managed to counteract the JD effect. Indeed, the Ci decreased at the value of 0.64. In contrast, SHR-SP exhibited a CIV that caused a very low control over aerobic NADH oxidation (Ci= 0.49). Consistently, the threshold plot showed an evident breakage. To sum up, on spontaneously hypertensive rats strains the saline diet can modify the kinetic of metabolic flux control analysis.

[1] S. Rubattu, S. Di Castro, H. Schulz, A.M. Geurts, M. Cotugno, F. Bianchi, H. Maatz, O. Hummel, S. Falak, R. Stanzione, S. Marchitti, S. Scarpino, B. Giusti, A. Kura, G.F. Gensini, F. Peyvandi, P.M. Mannucci, M. Rasura, S. Sciarretta, M.R. Dwinell, N. Hubner, M. Volpe, Ndufc2 Gene Inhibition Is Associated With Mitochondrial Dysfunction and Increased Stroke Susceptibility in an Animal Model of Complex Human Disease, Journal of the American Heart Association 5 (2016).

[2] S. Nesci, C. Algieri, F. Trombetti, M. Fabbri, G. Lenaz, Two separate pathways underlie NADH and succinate oxidation in swine heart mitochondria: Kinetic evidence on the mobile electron carriers, Biochimica et Biophysica Acta (BBA) - Bioenergetics (2023) 148977.

Cytochrome c oxidase (CytOx), the complex IV of the mitochondrial respiratory chain, is the oxygen accepting and rate-limiting enzyme of oxidative phosphorylation (OXPHOS) and a key player in the regulation of ATP supply. Recently, the allosteric ATP inhibition of CytOx was identified to control its activity based on the cellular energy status. In particular, when ATP levels are high and energy demands are met, ATP molecules bind to specific sites on CytOx, reducing the enzyme’s oxygen consumption, thereby conserving energy and maintaining the cellular homeostasis.

In healthy conditions, this mechanism ensures efficient energy production and prevents excessive generation of reactive oxygen species (ROS). However, in disease or stress conditions, allosteric ATP inhibition can be disrupted leading to increased ROS production, mitochondrial dysfunction, and cellular damage, contributing to various diseases such as neurodegenerative disorders, metabolic syndrome, and cardiovascular diseases.

In this study we analyzed how amino acid metabolism impacts the allosteric ATP inhibition of CytOx, given that amino acid levels and their metabolites can modulate the cellular ATP/ADP ratio and redox state, both of which are pivotal factors influencing CytOx activity.

Hence, we evaluated the "allosteric ATP-inhibition" of CytOx using intact isolated rat heart mitochondria. Under sequential injections of cytochrome c, to support the transfer of electrons between complex III and IV with molecular oxygen (O₂) serving as the final electron acceptor to produce water, we monitored oxygen consumption rates (OCR) as an indirect parameter for the CytOx activity. Using techniques such as the Seahorse extracellular flux analyzer, Oroboros Oxygraph or Clark-type oxygen electrodes, the decrease in oxygen concentration indicated decreased CytOx activity.

We identified certain amino acids, such as glutamate and aspartate, participating in pathways contributing to ATP-dependent inhibition of CytOx. This was in line with the inhibitory role of asparagin, a metabolite derived from aspartate and glutamine, pointing out a special role of glutamine metabolism and tricarboxylic acid (TCA) cycle in the process of allosteric ATP-inhibition. Following the OCR and CytOx activity assessment, further analysis of TCA cycle- and glutamine-derived mitochondrial metabolites by mass spectrometry supported our findings.

Overall, our results highlight a novel role of amino acid metabolism in regulating cellular energy homeostasis, beyond its traditional function in fueling TCA cycle anaplerosis. Understanding the regulatory mechanisms of CytOx allosteric ATP inhibition therefore improves our comprehension of cellular metabolism and offers promising potential as a therapeutic target for diseases associated with mitochondrial dysfunction.

Staphylococcus aureus and Pseudomonas aeruginosa are opportunistic pathogens, and have become major public health concerns because of the increased incidence of their drug resistance. The two organisms are responsible for both hospital and community-acquired infections.[1] [2]

S. aureus, a Gram-positive bacterium and P. aeruginosa, Gram-negative bacterium, are both facultative anaerobes that present a great ability to adapt to diverse environmental conditions, especially during host colonization, which makes them exceptional opportunistic pathogens. The adaptability of these pathogens comes from their metabolic versatility, which in part is due to the vast array of quinone reductases that connect different metabolic pathways to the respiratory chain.

Importantly the two bacteria produce quinones with different structures, S. aureus synthesizes menaquinone, a two ring-systems quinone, while P. aeruginosa produces ubiquinone, a one ring system quinone. Most relevant, P. aeruginosa also produces 2-Heptyl-4-hydroxyquinoline-N-oxide (HQNO), a quinone analogue that works as an inhibitor of most quinone interacting proteins and which confers to this bacterium a competitive advantage when colonizing the same niches with other bacteria, as the case of S. aureus. [3]

G3PQOs, Glycerol-3-phosphate:quinone oxidoreductases are flavin-containing monotopic enzymes, meaning they are attached to a single side of the lipid membrane, and catalyze the oxidation of glycerol-3-phosphate to dihydroxyacetone phosphate and concomitant reduction of quinone to quinol.[4]

In this work, we performed a comparative analysis of the G3PQOs from S. aureus and from P. aeruginosa. The expression and purification of G3PQOs was achieved and successful initial biochemical characterization of G3PQOs were performed. We observed that the enzyme from S. aureus is inhibited by HQNO, whereas that from P. aeruginosa is insensitive to this inhibitor.

References:

[1] S. Y. C. Tong, J. S. Davis, E. Eichenberger, T. L. Holland, and V. G. Fowler, “Staphylococcus aureus infections: Epidemiology, pathophysiology, clinical manifestations, and management,” Clin. Microbiol. Rev., vol. 28, no. 3, pp. 603–661, 2015, doi: 10.1128/CMR.00134-14.

[2] J. A. Driscoll, S. L. Brody, and M. H. Kollef, “The epidemiology, pathogenesis and treatment of Pseudomonas aeruginosa infections,” Drugs, vol. 67, no. 3, pp. 351–368, 2007, doi: 10.2165/00003495-200767030-00003.

[3] L. Biswas and F. Götz, “Molecular Mechanisms of Staphylococcus and Pseudomonas Interactions in Cystic Fibrosis,” Front. Cell. Infect. Microbiol., vol. 11, no. January, pp. 1–13, 2022, doi: 10.3389/fcimb.2021.824042.

[4] J. B. Daniels, J. Scoffield, J. L. Woolnough, and L. Silo-Suh, “Impact of glycerol-3-phosphate dehydrogenase on virulence factor production by Pseudomonas aeruginosa,” Can. J. Microbiol., vol. 60, no. 12, pp. 857–863, 2014, doi: 10.1139/cjm-2014-0485.

The penultimate of the five complexes in the electron transport chain is the terminal oxidase (complex IV) which couples the reduction of oxygen, and oxidation of cytochrome c or a quinol molecule creating a proton motive force. Cytochrome oxidases are diverse and their classification divides them into two main classes, the heme copper oxidases, and bd oxidases [1].

The native state of the Paracoccus cytochrome c oxidase, a heme copper oxidase has been discussed for many decades now. There is no direct evidence from UV visible spectroscopy yet to assign different states of the protein. Pinakoulaki and Proshlyakov have also performed Raman spectroscopic studies to predict the intermediates of the oxygen reaction mechanisms in different organisms. Hartmut Michel studied by UV visible spectroscopy the reaction of the native Paracoccus cytochrome c oxidase with non-saturating and saturating levels of hydrogen peroxide at different pH values predicting different artificial states of the protein. Recently, he showed some evidence from Cryo-EM studies the native state of the protein could be in a peroxide-bound state [2]. However, Kitagawa earlier showed the same with the bovine cytochrome c oxidase [3]. Furthermore, there are also spectroscopic differences between the recombinant one and the so-called ATCC form that is typically expressed in the organism.

Our UV/visible and Raman spectroscopic results show more insights into the native state of the protein. We also identified different artificial states of the oxygen reaction mechanism concerning the wild type recombinant and ATCC forms and compared it with the existing mechanisms.

[1] Nikolaev, A., Safarian, S., Thesseling, A., Wohlwend, D., Friedrich, T., Michel, H., Kusumoto, T., Sakamoto, J., Melin, F., & Hellwig, P., Electrocatalytic evidence of the diversity of the oxygen reaction in the bacterial bd oxidase from different organisms, BBA - Bioenergetics, (2021) 1862(8), 148436.

[2] Kolbe, F., Safarian, S., Piórek, Ż., Welsch, S., Müller, H., & Michel, H., Cryo-EM structures of intermediates suggest an alternative catalytic reaction cycle for cytochrome c oxidase, Nature Communications, (2021) 12(1), 6903.

[3] Ogura, T., & Kitagawa, T., Resonance Raman characterization of the P intermediate in the reaction of bovine cytochrome c oxidase. BBA - Bioenergetics, (2004) 1655, 290–297.

NADH:ubiquinone oxidoreductase, respiratory complex I, is a key enzyme in cellular respiration, coupling electron transfer from NADH to ubiquinone (Q) with proton translocating across the membrane. The Escherichia coli complex consist of 13 subunits, arranged in an L-shaped structure [1]. While electron transfer is reasonably well understood, the proton pumping mechanism remains still elusive. Based on molecular dynamics (MD) simulations it was proposed that an electric wave travels along the membrane arm leading to proton uptake from the N-side in the forward mode and proton release to the P-side in the backward mode. There, protons are expected to be translocated through each antiporter like subunit (NuoM, N and L) and the E-channel (NuoA, H, J and K) [3]. Based on structural analysis, the ”ND5-only” theory proposes two electric waves caused by the quinone chemistry and proton uptake from the N-side by NuoL and M and proton release to the P-side exclusively via NuoL (ND5 in mitochondrial complex I) [4].

Residues F408^M and F396^N are located in homologous positions at the exit of the putative proton pathways of NuoM and NuoN, thought to block proton “exit” in the framework of the “ND5 only” theory. Replacement of these phenylalanine residues by an aspartic acid residue significantly influenced the electron transfer and proton translocation activity of E. coli complex I.

Literature:

[1] T. Friedrich, B. Böttcher, The gross structure of the respiratory complex I: a Lego System,

BBA - Bioenergetics, 1608 (2004) 1-9.

[2] R. Baradaran, J. Berrisford, G. Minhas, L. A. Sazanov, Crystal structure of the entire respiratory complex I. Nature, 494 (2013) 443–448.

[3] V. R. I. Kaila, Long-range proton-coupled electron transfer in biological energy conversion: towards mechanistic understanding of respiratory complex I, J. R. Soc. Interface, 15 (2018) 144.

[4] D. Kampjut, L. A. Sazanov, Structure of respiratory complex I - An emerging blueprint for the mechanism. Curr. Opin. Struct. Biol., 74 (2022) 102350.

Molecular function of respiratory complex I (NADH:ubiquinone oxidoreductase) has remained one of the most controversial problems in bioenergetics. This large molecular machine (up to 1 MDa in some organisms) utilises the energy from NADH oxidation and quinone reduction for the pumping of four protons across the inner mitochondrial membrane. Being spatially separated by ca. 200 Å, it is not completely understood how these charge transfer processes are coupled together [1]. In our work, we address this question by employing microsecond-long classical molecular dynamics (MD) in combination with state-of-the-art hybrid quantum-mechanical/molecular-mechanical (QM/MM) free energy calculation methods.

Our simulations on the high-resolution structures of respiratory complex I from yeast [2], and mammalian species [3,4] reveal the mechanistic details of the long-range charge transfer processes in various catalytic regions of the enzyme. Particularly, we show that the protons can travel along the membrane-bound subunits parallel to the membrane [5], coupling the redox reaction to the proton pumping site(s). Also, we underline the importance of the partially reduced and partially protonated quinone species in the catalytic cycle of respiratory complex I, and how electron transfer between two quinone ligands can facilitate redox catalysis in certain physiological conditions [4].

Our findings challenge the current models of redox-coupled proton transfer by respiratory complex I and have far-reaching implications in understanding complex I-associated metabolic disorders on the atomistic scale.

[1] A.Djurabekova et al., Biochem J (2024) 481 (7): 499–514.

[2] K. Parey, J. Lasham et al., Sci. Adv. 7.46 (2021): eabj3221.

[3] J. Gu et al., Nat. Struct. Mol. Biol 29.2 (2022): 172-182.

[4] Y.-C. Shin, bioRxiv (2024)

[5] O.Zdorevskyi et al., Chem. Sci. 14.23 (2023): 6309-6318.

The Gram-negative multidrug resistant (MDR) pathogen Pseudomonas aeruginosa (Pa) can produce antibiotic-inactivating enzymes or form biofilm in the lungs of infected patients with cystic fibrosis (CF), causing chronic infections. Recent studies reported that the inhibition of enzymes involved in hydrogen sulfide (H₂S) metabolism leads to deficiency in Pa biofilm formation and antibiotic resistance [1], though contrasting results were also reported [2], making the role of H₂S in Pa physiology still debated.

Sulfide:quinone oxidoreductase (SQR) converts H₂S into glutathione persulfide (GSSH), using reduced glutathione (GSH) as a sulfur acceptor and transferring electron equivalents to coenzyme Q. Here, we characterized from a biochemical viewpoint the isoform 2 of PaSQR, as recombinantly produced in Escherichia coli. The enzyme was purified by affinity chromatography, and its oligomeric state analyzed using size-exclusion chromatography. Static and time-resolved fluorescence, far-UV circular dichroism and UV-vis absorption spectroscopies were used to investigate structural and functional properties of PaSQR. The enzyme catalytic activity was measured following quinone reduction by UV-vis absorption spectroscopy. Our findings suggest that PaSQR contributes to keep H₂S concentration low, thus possibly protecting Pa from H₂S poisoning. Further studies on the structure and regulation of PaSQR catalytic activity will help to better understand the role of this protein in MDR pathogens.

References

[1] K. Shatalin, A. Nuthanakanti, P. Fedichev, A. Serganov, E. Nudler, Inhibitors of bacterial H₂S biogenesis targeting antibiotic resistance and tolerance, Science 372(2021)1169-1175.

[2] L. Caruso, M. Mellini, O. Catalano Gonzaga, A. Astegno, E. Forte, A. Di Matteo, A. Giuffrè, P. Visca, F. Imperi, L. Leoni, G. Rampioni, Hydrogen sulfide production does not affect antibiotic resistance in Pseudomonas aeruginosa, Antimicrob Agents Chemother., 68 (2024) e0007524

NADH:ubiquinone oxidoreductase, respiratory I, is a key enzyme in cellular energy metabolism. It couples electron transfer from NADH to ubiquinone in its peripheral arm with proton translocation across the membrane in its membrane arm. The coupling of these two processes remains, however, elusive [1]. Is is generally accepted that the quinone redox chemistry drives proton translocation by a yet unknown mechanism. Quinone is reduced in a unique cavity between the two arms [2]. It was proposed that a reduced quinone species is protonated by conserved amino acid residuesof the cavity. However, site-directed mutagenesis indicates that this is not the case. Furthermore, redox-difference UV/vis spectra from Escherichia coli complex I indicated the formation of a quinol anion during turn-over [3, 4]. Here, a mechanism for energy conversion including a quinol anion as catalytic intermediate is proposed.

[1] I. Chung, D. N. Grba, J. J. Wright, J. Hirst, Making the leap from structure to mechanism: are the open states of mammalian complex I identified by cryoEM resting states or catalytic intermediates?, Curr. Opin. Struct. Biol., 77 (2022) 102447.

[2] K. Fiedorczuk, J. A. Letts, G. Degliesposti, K. Kaszuba, M. Skehel, L. A. Sazanov, Atomic structure of the entire mammalian mitochondrial complex I, Nature, 538 (2016) 406-410.

[3] F. Nuber, L. Mérono, S. Oppermann, J. Schimpf, D. Wohlwend, T. Friedrich, A Quinol Anion as Catalytic Intermediate Coupling Proton Translocation with Electron Transfer in E. coli Respiratory Complex I, Front. Chem., 9 (2021) 672969.

[3] V. R. Kaila, Resolving Chemical Dynamics in Biological Energy Conversion: Long-range Proton-Coupled Electron transfer in Respiratory Complex I, Acc. Chem. Res., 54 (2021) 4462-4473.

The NADH:ubiquinone oxidoreductase (complex I) is the first enzyme of the respiratory chain that oxidizes NADH and reduces quinone coupling this reaction with the translocation of protons across the membrane. The enzyme is L-shaped with a membrane domain and peripheral arm. Proton translocation takes place in the membrane domain, the oxidoreductase activity in the peripheral arm. NADH is oxidized by transferring two electrons to FMN and from there over the iron-sulfur clusters N3, N1b, N4, N5, N6a, N6b and N2 to ubiquinone [1]. It is generally accepted that N2 has to be reduced by NADH due to its positive redox potential. However, recent EPR studies in our lab have shown that N2 is not reduced, if a quinone is not available in sufficient quantities.

[1] Gnandt E, Dörner K, Strampraad MFJ, de Vries S, Friedrich T. The multitude of iron-sulfur clusters in respiratory complex I. Biochim Biophys Acta. 2016 Aug; 1857(8):1068-1072. doi: 10.1016/j.bbabio.2016.02.018. Epub 2016 Mar 2. PMID: 26944855.

Respiratory complex I, NADH:ubiquinone oxidoreductase, plays a central role in energy metabolism by coupling NADH oxidation to proton translocation. Its dysfunction is associated with degenerative diseases in humans through an enhanced production of reactive oxygen species (ROS). The mechanism of ROS formation is not yet fully understood, but it is generally accepted that they are formed by the redox chemistry of the flavin mononucleotide (FMN) cofactor. It was proposed that FMN can reversibly dissociate from complex I, halting the transfer of electrons and thus decreasing ROS production. However, this is not supported by X-ray data, showing that the FMN is bound with full occupancy and low B-factors in both, the oxidized and reduced state of NuoEF, the electron input module of complex I, and experiences no change in binding by the oxidation state [1]. Here, the redox potential of FMN in the presence of NADH was determined in complex I and NuoEF by electrochemical titration. Free FMN has a redox potential of -207 mV that is shifted to -330 mV and -350 mV in complex I of Aquifex aeolicus and Escherichia coli, respectively [2]. In presence of NADH, the FMN redox potential is shifted to more positive values, leading to thermodynamically stronger binding of FMN than previously assumed. Furthermore, the role of Fe/S cluster N1a was investigated. Mitochondrial complex I produces predominantly superoxide, while E. coli complex I forms H₂O₂. It was speculated that this difference arises from the more negative redox potential of the nearby Fe/S cluster N1a. Altering the redox potential of N1a by site-directed mutagenesis did not alter the produced ROS species [3]. Here, it is shown that the redox potential of N1a influences that of FMN.

Literature:

[1] M. Schulte et al., A mechanism to prevent production of reactive oxygen species by Escherichia coli respiratory complex I, Nat. Commun. 10 (2019) 2551.

[2] S.G. Mayhew, The effects of pH and semiquinone formation on the oxidation-reduction potentials of flavin mononucleotide. A reappraisal, Eur. J. Biochem. 265 (1999) 698-702.

[3] J.A. Birrell et al., Investigating the function of 2Fe-2S cluster N1a, the off-pathway cluster in complex I, by manipulating its reduction potential, Biochem. J. 456 (2013) 139–146.

F_oF₁-ATPase is one of the main enzymes in bacteria that helps to maintain cell viability and provides the cell energy under fermentative conditions [1]. Propionic acid (PPA) is considered as an inducer for autism spectrum disorders and has a significant impact on cell viability and bioenergetic properties [2] [3]. The impact of different concentrations of PPA (11.7 and 33.4mM) on proton ATPase activity was investigated in Escherichia coli strains isolated from the gut of healthy and autism like behavior exhibiting rats. ATPase activity was determined by the amount of inorganic phosphate (P_i) produced in the reaction of membrane vesicles with ATP. The ATPase activity of the strain isolated from the cecum of healthy rat was ~ 32 nmol P_i(min μg protein)⁻¹. Interestingly, the addition of PPA and K⁺ ions had no significant effect on ATPase activity. The overall ATPase activity in the strain from the PPA injected rat’s cecum was ~ 30 nmol P_i (min μg protein)⁻¹. The addition of 11.7 mM of PPA reduced the activity by ~ 2.6 fold. It is suggested that cells reduce ATPase activity to use the ATP energy for their growth and adaptation to acid presence. On the other hand, the presence of K⁺ ions and 11.7 mM PPA stimulate the ATPase activity by ~ 3.6 fold compared to its control. Similar pattern was observed in the strain isolated from the colon of PPA injected rat. The K⁺ ions and the presence of 11.7 mM PPA increased ATPase activity by ~ 1.6 fold. However, the strains isolated from the small intestine ATPase activity stimulation by K⁺ ions was not observed. Furthermore, this pattern is only notable in the 11.7 mM concentrations of PPA, while the addition of 33.4 mM PPA does not significantly affect ATPase activity. The obtained results highlight the ATPase regulating role of K⁺ ions, which is used by strains isolated from the PPA injected rat’s cecum and colon to maintain cell homeostasis and adaptation to the PPA environment.

[1] A. Trchounian, K. Trchounian, Fermentation Revisited: How Do Microorganisms Survive Under Energy-Limited Conditions?, Trends Biochem Sci, 44 (2019) 391–400, https://doi.org/10.1016/j.tibs.2018.12.009.

[2] R. Paudel, K. Raj, Y.K. Gupta, S. Singh, Oxiracetam and Zinc Ameliorates Autism-Like Symptoms in Propionic Acid Model of Rats, Neurotox Res, 37 (2020) 815–826, https://doi.org/10.1007/s12640-020-00169-1.

[3] H. Gevorgyan, T. Abaghyan, M. Mirumyan, K. Yenkoyan, K. Trchounian, Propionic and valproic acids have an impact on bacteria viability, proton flux and ATPase activity, J Bioenerg Biomembr, 55 (2023) 397–408, https://doi.org/10.1007/s10863-023-09983-6.

The mitochondrial ATP synthase uses the proton motive force generated by the electron transport chain to synthetize ATP in the process of oxidative phosphorylation. The reaction is reversible whereby ATP is hydrolysed and protons are pumped across the inner mitochondrial membrane (IMM) into the intra-cristae space. In mammals, dimers of ATP synthase are located at the edges of the cristae forming arrays that shape the lamellar sheets of cristae membranes [1]. Thereby, inhibitory factor 1 (IF1) controls ATP synthase activity and organization. IF1 intercalates with ATP synthase by blocking the reverse rotation and ATP hydrolysis. Overexpression of IF1 results in changes in IMM ultrastructure with increased fusion of cristae. These ultrastructural changes in turn affects the spatiotemporal organization of ATP synthase and suggest that the oligomerization state of ATP synthase is crucial for optimal ATP synthesis [2].

To better understand the effects of the supramolecular organization of ATP synthase at molecular and enzymatic level, the supramolecular assembly of ATP synthase will be altered without directly interfering with enzyme activity. IF1 also bridges neighbouring mitochondrial ATP synthase dimers into tetramers [3]. Likewise, the subunit DAPIT (diabetes-associated protein in insulin-sensitive tissues) of ATP synthase promotes dimer-dimer stability similar to IF1 [4]. By manipulating DAPIT levels, the effects of the supramolecular organization of ATP synthase on its activity will be investigated without directly affecting its function.

[1] T. B. Blum, A. Hahn, T. Meier, K. M. Davies, W. Kuhlbrandt, Dimers of mitochondrial ATP synthase induce membrane curvature and self-assemble into rows, PNAS, 116 (2019) 4250-4255.

[2] B. Rieger, T. Arroum, M. T. Borowski, J. Villalta, K. B. Busch, Mitochondrial F1 FO ATP synthase determines the local proton motive force at cristae rims, EMBO Rep, 22 (2021) e52727.

[3] J. Gu, L. Zhang, S. Zong, R. Guo, T. Liu, J. Yi, P. Wang, W. Zhuo, M. Yang, Cryo-EM structure of the mammalian ATP synthase tetramer bound with inhibitory protein IF1, Science, 364 (2019) 1068-1075.

[4] J. He, H. C. Ford, J. Carroll, C. Douglas, E. Gonzales, S. Ding, I. M. Fearnley, J. E. Walker, Assembly of the membrane domain of ATP synthase in human mitochondria, PNAS, 115 (2018) 2988-2993.

The Permeability Transition (PT) is a mitochondrial permeability increase to ions and solutes caused by the opening of a Ca²⁺ and voltage-dependent channel, the PT pore (PTP). The long-standing debate about the PTP molecular identity has been recently challenged by a substantial set of data reporting a key role of the ATP synthase. In presence of Ca²⁺_, the enzyme generates high conductance channels with biophysical properties matching those of the PTP. However, the mechanism of channel formation by the ATP synthase is still elusive. A central role is played by the dimerization subunits (e and g), which are required for the assembly of higher-order structures. In the absence of e and g subunits, the ATP synthase can no longer generate a channel, and a PT can eventually be mediated by the adenine nucleotide translocator (ANT), which represents an alternative permeation pathway [1]. High-resolution structures of the ATP synthase revealed a physical connection between the e subunit C-terminus and the lipids filling the rotor (c-ring) and showed Ca²⁺-dependent conformational changes that dramatically perturb this region [2]. One hypothesis is that, in presence of Ca²⁺, the e subunit C-terminus removes the lipids from the c-ring via a push-pull mechanism leading to the channel formation within the rotor. To test this, we generated HeLa cells expressing a truncated form of subunit e devoid of its C-terminus and evaluated PT occurrence. Our data show that in the mutant cells the PT is mediated by ANT, indicating that the e subunit C-terminus is an indispensable requirement for the channel formation by the ATP synthase.

[1] A. Carrer, L. Tommasin, J. Šileikytė, F. Ciscato, R. Filadi, A. Urbani, M. Forte, A. Rasola, I. Szabò, M. Carraro, P. Bernardi, Defining the molecular mechanisms of the mitochondrial permeability transition through genetic manipulation of F-ATP synthase, Nature Communications. 12 (2021) 4835–4835.

[2] G. Pinke, L. Zhou, L.A. Sazanov, Cryo-EM structure of the entire mammalian F-type ATP synthase, Nature Structural and Molecular Biology. 27 (2020) 1077–1085.

The ubiquity of adenosine triphosphate (ATP) as the universal energy currency across almost all species underscores its pivotal role in facilitating life. For mesophilic organisms, steady-state ATP levels increase with temperature as one would expect from the Arrhenius relationship. Surprisingly, psychrophiles from all domains of life show an inverse relationship where cellular ATP levels increase as temperatures drop. Though the mechanism underlying this phenomenon is unknown, increased ATP levels likely contribute to psychrophily in these organisms by increasing the probability of molecular collisions with ATP—the universal currency of energy—under conditions of reduced molecular motion (e.g., low temperature). We sequenced the transcriptome of the glacial ice worm Mesenchytraeus solifugus, which elevates ATP almost 2-fold over a ~10˚C temperature drop and identified an 18 residue C-terminal extension of the mitochondrially encoded ATP6 subunit of the ATP synthase complex, which forms the proton pore that drives ATP synthesis. This sequence is not found on any other ATP6 protein in GenBank but is present on a variety of bacterial ion channels. Since manipulation of this mitochondrially-encoded gene is impossible in M. solifugus, we investigated the impact of this extension by making a chromosomal knock-in of this fusion into the homologous gene atpB in E. coli (exAtpB). Expression of exAtpB significantly increased the Vmax of the ATP synthase complex without affecting Km. Remarkably, this enhanced activity persisted in the bacterium Caulobacter crescentus despite structural differences in the ATP synthase complex. The broad efficacy of this C-terminal extension across species underscores its potential as a simple yet potent modulator of ATP synthase catalytic efficiency.

The F₁-ATPase rotates subunit-γ in 120° power strokes within its ring of three catalytic sites where 0° is defined as the start of a rotary power stroke. Energy to rotate subunit-γ derives from ATP-binding even though rotation is interrupted ~85° after ATP binds by the catalytic dwell when ATP hydrolysis and Pi release occurs. How the energy from ATP binding is used to hydrolyze ATP and release Pi during the catalytic dwell, which subsequently enables subunit-γ to rotate ~35° when the next ATP binds is a major unresolved question. By monitoring subunit-γ rotary position every 5 μs, we resolved Catalytic dwell oscillations that are initially centered at 0° and extend CCW to 13° and CW to -13°, which is consistent with F₁ structures containing bound transition state inhibitors. These oscillations, which decay by a first order process consistent with ATP hydrolysis, are replaced by oscillations that, 4 of 5 times, are centered at 14° and extend from 3° and 25°. The remaining 1 of 5 oscillations are centered at 33° that extend by as much as 27° and 44°, which is comparable to the rotary position of ATP binding. Remarkably, the rotary position returns to 0° at the end of the Catalytic dwell. This keeps the starting point of power strokes in phase with the rotational position of subunit-γ over the course of hundreds of consecutive rotational events. The observed oscillations correlate with F₁ structures with catalytic site occupancies and the rotary position of subunit-γ that previously appeared to be inconsistent with the alternating site mechanism. These results provide new insight into the ATPase-dependent rotation mechanism that indicates a ratchet mechanism in which subunit-γ oscillations enable changes in catalytic site occupancy that continue until the three catalytic sites contain the correct compliment of substrates and products to initiate the subsequent power stroke that is biased to rotate counter-clockwise.

Mitochondria are a target site of heavy metals poisoning. The heavy metals alter many mitochondrial functions such as the inner membrane permeability, the membrane potential, uncouple oxygen uptake and ATP synthesis, and increase ROS production. These alterations compromise cell survival. Although there are many evidences about mitochondrial malfunction during heavy metals intoxication, few is knowing about the direct effect of heavy metals on the activity of OXPHOS complexes. In this work the monomer and dimer of the F₁F_O-ATP synthase were solubilized with digitonin and isolated in their active form, and the effect of As³, Cd²⁺, Cu²⁺, Hg²⁺ and Zn²⁺ on the ATPase activity of these oligomers was quantified. The monomer was inhibited by Hg²⁺ (IC₅₀ = 2 mM), As³⁺ (IC₅₀ = 10 mM), Cu²⁺ (IC₅₀ = 23 mM), and Cd²⁺ (IC₅₀ = 76 mM) while Zn²⁺ have not effect on monomer activity. The dimer was inhibited only by Hg²⁺ (IC₅₀ = 11 mM) and Cu²⁺ (IC₅₀ = 74 mM), and was resistant to As³⁺, Zn²⁺, and Cd²⁺. The IC₅₀ values are physiological relevant and are comparable to known level of heavy metals poisoning. The ATPase activity of the monomer and dimer was reactivated by dilution of the heavy metals, indicating that inhibition occurs without chemical modification of the F₁F_O-ATP synthase. Our current data provides novel important insight into the differential effect of the heavy metals on the dimer and monomer of the F₁F_O-ATP synthase. In the context of the F₁F_O-ATP synthase, a single structural difference between the monomer and dimer is the presence of a dimerization zone within the hydrophobic sector. Our findings indicate that this dissimilarity may stem from interactions within this region. Specifically, the dimer configuration offers a unique angle and incorporates specific dimerized subunits absent in the monomer; particularly, the dimerizing subunit f is occluding the proton access to the hemi-channel of the F_O-sector. This architectural difference (i.e. stereochemical hindrance) between the monomer and the dimer could explain the dimer resistance against As³⁺ and Cd²⁺.

This work was supported by PAPIIT (IN201923) from Universidad Nacional Autónoma de México (UNAM). GGC (1103534) is a PhD student of the Posgrado en Ciencias Biomédicas from UNAM and CONACyT fellow.

Mitochondrial ATP synthases are multi-subunit transmembrane protein complexes, localized in the inner mitochondrial membrane. In eukaryotic cells, the ATP synthases are responsible for ATP production during respiration; nevertheless, the enzyme is also considered to form the nonselective channel named permeability transition pore (PTP). The pore is formed in response to elevated Ca²⁺ concentration and is facilitated by oxidative stress and ATP depletion. Despite the permeability transition being described from the physiological point of view for major groups of eucaryotes - animals, fungi, and higher plants – the structural features of the PTP remain unknown [1]. Mitochondrial permeability transition mediates ischemia-reperfusion injury in mammals and plant cell death caused by oxidative stress, therefore knowledge of PTP molecular structure can provide insights into how to counteract these phenomena [2, 3].

In this work, we aimed to obtain conformational changes in murine mitochondrial ATP synthase induced with Ca²⁺. We collected and processed two cryo-EM datasets - one with native enzyme and the other with ATP synthase exposed to 1 mM Ca²⁺. In the Ca²⁺ dataset, we identified conformational changes that may be associated with the reorganization of ATP synthase into PTP.

[1] E. Frigo, L. Tommasin, G. Lippe, M. Carraro, P. Bernardi, The Haves and Have-Nots: The Mitochondrial Permeability Transition Pore across Species, Cells, 12(10) (2023) 1409.

[2] A.P. Halestrap, A.P. Richardson, The mitochondrial permeability transition: a current perspective on its identity and role in ischaemia/reperfusion injury, J Mol Cell Cardiol, 78 (2015) 129–141.

[3] B.S. Tiwari, B. Belenghi, A. Levine, Oxidative stress increased respiration and generation of reactive oxygen species, resulting in ATP depletion, opening of mitochondrial permeability transition, and programmed cell death, Plant physiol,128(4) (2002) 1271–1281.

ATeam fluorescent proteins are extensively utilized to monitor ATP concentrations within living cells due to their ability to reversibly bind ATP. This binding induces a conformational change, detectable through fluorometry.

We purified ATeam from bacteria and used it for in vitro measurements of ATPase activity of ATP-synthases from Escherichia coli, Bacillus subtilis, and thermophilic Bacillus sp. PS3 under conditions when ADP and phosphate are present. Traditional methods of ATPase activity measurements based on registration of phosphate release, on monitoring the acidification of the medium, or on regeneration of ADP to ATP coupled to NADH oxidation do not allow real-time measurements under such conditions. We found that the ATPase activity of Escherichia coli enzyme was significantly suppressed upon addition of ADP and phosphate. However, the enzymes from Bacillus subtilis and Bacillus sp. PS3 retained most of their ATPase activity. It was also found that the level of ADP-inhibition of Bacillus subtilis ATP synthase decreases with increasing temperature.

When expressed with a mitochondrial address in yeast cells, ATeam is accumulated in mitochondria. We purified mitochondria with ATeam and measured ATP synthesis and hydrolysis following the spectral changes of ATeam fluorescence.

In conclusion, ATeam proteins offer a practical and effective method for real-time analysis of ATP synthesis and hydrolysis both in vitro and in isolated mitochondria.

This study was funded by the Russian Scientific Foundation (Grant No. 20-14-00268).

Acinetobacter baumannii is regarded as one of the most antibiotic-resistant bacteria and belongs to the class of nonfermenting and strictly aerobic pathogen [1]. Due to this metabolic restriction, the pathogen is dependent on oxidative phosphorylation and the enzyme catalysing the synthesis of ADP and P_I to ATP, the F₁F_O-ATP synthase (α₃:ß₃:γ:δ:ε:a:b₂:c₁₀). Proton pumping through the a:c₁₀ subunits drives rotation of the γ:ε domain, which transfers the energy to the catalytic α₃:ß₃ headpiece [2]. In order to control the formed currency of life, ATP, the A. baumannii engine shows low ATP hydrolysis, preventing the pathogen from wasting energy [2, 3]. Subunit ε has been proposed to play a key role in regulating this latent ATP hydrolysis [2]. Here we present the cryo-electron microscopy structure of the recombinant A. baumannii F₁-ATPase at 3.0 Å resolution and decipher by mutagenesis studies the epitopes of the A. baumannii ε subunit involved in the inhibitory steps [3]. In addition, we will present new atomic structures of the enzyme, visualizing transition states of this regulatory subunit from its compact (non-inhibitory) to its extended (inhibitory) states.

[1] K. Ioannis, E. Vasileiou, Z. Dorothea Pana, and A. Tragiannidis, Acinetobacter Baumannii Antibiotic Resistance Mechanisms, Pathogens 10, no. 3 (2021): 373.

[2] J.K. Demmer, B.P. Phillips, O.L. Uhrig, A. Filloux, L.P. Allsopp, M. Bublitz, T. Meier, Structure of ATP synthase from ESKAPE pathogen Acinetobacter baumannii, Sci Adv, 8 (2022) eabl5966.

[3] W.-G. Saw, K.C.M. Le, J. Shin, J.H.M. Kwek, Chui F. Wong, P. Ragunathan, T.C. Fong, V. Müller, G. Grüber, Atomic insights of an up and down conformation of the Acinetobacter baumannii F₁-ATPase subunit ε and deciphering the residues critical for ATP hydrolysis inhibition and ATP synthesis, FASEB J, 37 (2023) e23040.

Mitochondrial ATP Synthase or Complex V (CV) is the main producer of Adenosine Triphosphate (ATP) for the cell. However, when respiratory function is impaired, CV can also function in reverse to hydrolyze ATP. Dimerization of CV facilitates the formation of invaginations in the mitochondrial inner membrane (IMM) called cristae, which respond to and maintain the proton motive force. The dynamics of the interaction between the hydrolysis of ATP by CV and cristae architecture maintenance are not fully understood. Here we show that impairment of cristae ultrastructure in mitochondrial contact site and cristae organizing system (MICOS) and OPA1 knockout cell models is linked to changes in mitochondrial CV ATP hydrolytic capacity. Moreover, we have found that the selective inhibitor of CV reverse activity (+)-Epicatechin can reduce hydrolytic capacity in these models, potentially rescuing the defects in mitochondrial architecture. Previous data has also shown that mitochondrial membrane potential is heterogenous, with cristae maintaining distinct electrochemical domains. We hereby propose that cristae architecture and CV ATP hydrolysis are linked by the dimerization of CV and maintenance of the proton motive force.

Plant extracts (PEs) and biologically synthesized nanoparticles (NPs) serve as sources of biologically active compounds. Among these, compounds with antibacterial properties have a special value in light of the current challenges due to problems with antibiotic resistance. The high therapeutic value of plant-based compounds, their synergistic abilities with known antibiotics, and their limited side effects are advantages. F_OF₁-ATPase/synthase is one of the primary enzymes of bioenergetic relevance in microbes, which can serve as a potential target for antimicrobials. Armenian flora is rich with plant species exhibiting antimicrobial properties against pathogenic, non-pathogenic, antibiotic-susceptible, and resistant microbial strains. In the study, ethanol extracts of Ribes rubrum, Rumex obtusifolius, Hypericum alpestre, Origanum vulgare, Teucrium polium, Alkanna orientalis are shown to suppress the growth of Escherichia coli ATCC25922, kanamycin-resistant E. coli pARG-25, ampicillin-resistant E. coli DH5α-pUC18, Salmonella typhimurium MDC1754, Enterococcus hirae ATCC9790, Bacilllus subtilis A1. The chemical composition of plant extracts was investigated using HPLC-MS and GC-MS methods. In the presence of plant-borne compounds, the µ value of E. coli decreased up to 2.5 times with an increase of G value. Moreover, the influence of plant extracts on the F_OF₁-ATPase activity and energy-dependent H⁺-fluxes in E. hirae and E. coli antibiotic-resistant strains were shown. The antibacterial effect of silver NPs on various bacteria (E. coli BW25113, S. typhimurium MDC1759, E. hirae ATCC9790, and Staphylococcus aureus MDC5233), including drug-resistant E. coli strains, was shown. The antimicrobial action of NPs was shown to result from their interaction with bacterial F_OF₁-ATPase. A green synthesis of silver NPs using the medicinal Artemisia annua L. and Stevia rebaudiana also demonstrated antibacterial activity.

The data obtained through this study deepens our basic understanding of the role of cell membranes, particularly, F_OF₁-ATPase/synthase, redox status in cells, and will open up new possibilities to address the problem of antibiotic resistance. The results contribute to the global search for new antimicrobials.

The F1Fo ATP synthase catalyses the production of the universal cellular energy currency ATP empowered by an electrochemical gradient across a biological membrane. There are previously characterised compounds which specifically interact with the ATP synthase and inhibit its enzymatic function, as it was exemplarily shown for oligomycin. Another compound which seems to interact specifically with the mitochondrial ATP synthase is termed leucinostatin A, a nature-derived hydrophobic oligopeptide. It has been reported that leucinostatin A has a dual inhibitory effect on oxidative phosphorylation. At lower concentrations (< 240 nM), it is proposed that leucinostatin A interacts specifically with the ATP synthase by binding to the membrane-embedded Fo-part of the enzyme. At higher concentrations (> 300 nM), leucinostatin A acts as a protonophoric uncoupler of the mitochondrial membrane potential.

In this project, we unambiguously identified leucinostatin A as a highly potent mitochondrial ATP synthase inhibitor of various organisms. Our data indicate a specific binding mechanism to the membrane-embedded moiety of the ATP synthase that involves the proton binding motif. In a structure-activity relationship (SAR) including newly synthesized leucinostatin A derivatives, we identified the hydroxyleucine located at position 7 in leucinostatin A to be the responsible moiety for ATP synthase inhibition. Using high-resolution respirometry and a liposome-based ATP producing system, we provide the detailed molecular explanation for leucinostatin A toxicity in mammals.

ATP synthase is a key enzyme in mitochondrial bioenergetics. Typically, it utilizes the energy of transmembrane proton potential difference, maintained by the respiratory chain, to synthesize ATP. However, under membrane de-energization, ATP synthase can hydrolyze ATP. This ATPase activity can significantly decrease intracellular ATP levels and thus requires strict regulation. A common regulatory mechanism is non-competitive inhibition of the enzyme ATPase activity by Mg-ADP (ADP-inhibition) [1]. Certain mutations are known to affect the extent of ADP-inhibition; one of them is 𝛾M23K, previously studied in Rhodobacter capsulatus, Bacillus sp. PS3 and Escherichia coli. In these organisms, the M23K mutation has been shown to enhance ADP-inhibition [2,3].

Here, we have obtained a yeast strain with 𝛾M23K mutation using the CRISPR-Cas system. Measurements of ATPase activity of isolated mitochondria and submitochondrial particles have shown the increased level of ADP-inhibition. The inhibitory effect of ADP added to the ATPase reaction was more pronounced in samples obtained from the mutant strain in comparison with a parent strain. When ATPase activity was measured in the ATP-regenerating system, the mutant demonstrated a long lag period after adding ATP in the sample, which may also mean that ADP-inhibition in the mutant enzyme is enhanced.

On a physiological level, the mutation resulted in a decrease in maximal growth rate (𝜇) both for Rho+ (with mtDNA) and Rho0 (without mtDNA) cells. However, if we used starved 7-day stationary cultures for inoculation, the mutant Rho0 strain had increased 𝜇 in comparison with wild-type Rho0 cells.

The work is supported by Russian Science Foundation grant 20-14-00268.

[1] Lapashina AS, Feniouk BA. ADP-Inhibition of H+-FF-ATP Synthase. Biochemistry (Moscow). 2018;83: 1141–1160.

[2] Bandyopadhyay S, Allison WS. GammaM23K, gammaM232K, and gammaL77K single substitutions in the TF1-ATPase lower ATPase activity by disrupting a cluster of hydrophobic side chains. Biochemistry. 2004;43: 9495–9501.

[3] Feniouk BA, Rebecchi A, Giovannini D, Anefors S, Mulkidjanian AY, Junge W, et al. Met23Lys mutation in subunit gamma of F(O)F(1)-ATP synthase from Rhodobacter capsulatus impairs the activation of ATP hydrolysis by protonmotive force. Biochim Biophys Acta. 2007;1767: 1319–1330.

Most mitochondrial proteins are nuclear-encoded; therefore, they must be imported into mitochondria to function properly. To successfully transport proteins to their respective mitochondrial sub-compartments, eukaryotes have evolved an intricate translocation system requiring a mitochondrial targeting sequence (MTS). The first entry pathway of proteins into mitochondria is through the translocate of the outer mitochondrial membrane (TOM). TOM core complex is a dimer with subunits consisting of the pore-forming subunit Tom40 and the structural and receptor subunits Tom5-7 and Tom22, respectively. Together with the core components, the two receptor subunits, Tom20 and Tom70, make up the holo complex. In previous work on TOM from Neurospora crassa we found that Tom20 is labile and assumes two conformations [Ornelas et al, 2023]. To mitigate against the loss of TOM subunits during purification, we genetically transformed the thermophilic eukaryote Chaetomium thermophilum to express Tom22 with an epitope tag. We isolated and purified thermostable Ct-TOM and determined its cryo-EM structure with and without bound MTS. As before, the core components of Ct-TOM are well defined. Around the complex and at the dimer interface we identified a number of ordered lipids, and we noted that the position of the lipid joining the two Tom40 pores is conserved. We observe two Tom20 receptors per TOM dimer in a range of conformations. In the predominant conformation the two Tom20 receptor domains interact closely above the pores. When Ct-TOM was supplemented with MTS, we observed clear corresponding densities bound in each of the two Tom40 pores. Our work provides new insights into the first steps of protein translocation into mitochondria.

Reference: P. Ornelas, T. Bausewein, J. Martin, N. Morgner, S. Nussberger, W. Kühlbrandt, Two conformations of the Tom20 preprotein receptor in the TOM holo complex, Proc. Natl. Acad. Sci. U. S. A., 120 (2023) e2301447120.

Escherichia coli encodes two formate channels, FocA and FocB, that either export formate or import depending on fermentable sugar and external pH [1].
The current study delves into the modulation of proton flux in Escherichia coli mutants exhibiting deficiencies in the FocA and FocB formate channels, examining their response during the utilization of mixed carbon sources. This investigation aims to elucidate how the interplay between these channels orchestrates formate transport, particularly in the context of fermentable sugars and external pH variations.
The bacteria were cultured on a peptone medium supplemented with glucose (11.1 mM), glycerol (137 mM), and formate (10 mM) at pH 7.5. The overall proton flux (ΔJH+) was quantified using a proton electrode. Throughout the experiment, glucose (11.1 mM) served as an intracellular formate source, while extracellular formate (10 mM) was introduced separately. To assess the involvement of F_OF₁-ATPase, bacterial cells were exposed to 0.2 mM N’N’-dicyclohexylcarbodiimide (DCCD), a specific inhibitor of F_OF₁-ATPase, under anaerobic conditions.
When glucose was supplemented total J_H+ was 2.2 mmol min^-1 per 10⁹ cells. In focA mutant J_H+ remained similar, meanwhile in single focB and double focAfocB mutants it increased by 40%. DCCD-sensitive J_H+ in all mutants similarly increased by ~50% upon glucose utilization. When formate was supplemented J_H+ was totally suppressed, in focB resulting in a 50% reduction, and in focAfocB mutant exhibited 0.12 mmol min^-1 similar J_H+ to wild type.
Taken together the results state that the function of formate channels in proton translocation is strongly differs depending on fermenting sugars, particularly here FocA had an important role in the transport of formic acid into the cell, meanwhile FocB is responsible mostly for formate efflux.

[1] Babayan A., Vassilian A., Poladyan A., Trchounian K, Role of the Escherichia coli FocA and FocB formate channels in controlling proton/potassium fluxes and hydrogen production during osmotic stress in energy-limited, stationary phase fermenting cells. Biochimie; 221:91-98. doi: 10.1016/j.biochi.2024.01.017

Kv1.3 is a potassium channel with high expression in neurons and immune cells in physiological conditions. Kv1.3 is located both to the plasma- and mitochondrial inner membranes. Its expression is increased in the microglia upon activation; in contrast, inhibition of Kv1.3 locks microglia in an anti-inflammatory polarized state. Therefore, Kv1.3 is considered a possible therapeutic target against Parkinson and Alzheimer diseases, where a chronic neuroinflammation due to activated microglia exacerbates neuronal cell death. In order to understand the specific role of Kv1.3 in microglia, we generated a tissue-specific Kv1.3 KO mouse model. Following the validation and characterization of this novel model, we exploited a well-studied Parkinson disease (PD) model where the neurotoxin MPTP was used in order to block complex I activity of the respiratory chain. We observed that lack of Kv1.3 in the microglia attenuated the loss of dopaminergic neurons, and led to maintenance of motor coordination ability. Our data indicate that Kv1.3 deletion/inhibition might be a promising approach to counteract respiratory chain complex I inhibition-induced PD.

SNAT2 is a Na+-dependent transporter of small and medium neutral amino acids [1]. It is ubiquitously expressed in human tissues and involved in pathologies such as cancer and type II diabetes. Despite its relevance, the lack of an “in vitro” assay for elucidating its structure/function relationship hampers the discovery of inhibitors/modulators for this key transporter. The full-length cDNA coding for the hSNAT2 transporter was cloned in the pET-28a-Mistic vector. The presence of the Mistic tag at the N-terminus of the protein, the addition of glucose to the growth medium, and a tightly controlled oxygenation were crucial for SNAT2 expression in the BL21(DE3) codon plus RIL strain. The over-expressed protein was purified by IMAC exploiting the 6His tag at the C-terminus with a yield of about 60 mg/Liter cell culture. The transport activity of the purified protein was assayed in proteoliposomes. The recombinant SNAT2 showed specificity towards amino acids and kinetic parameters similar to those previously described in intact cells [3]. Therefore, the in vitro transport assay can be used for testing ligands identified by vHTS [2].

[1] S. Broer, The SLC38 family of sodium-amino acid co-transporters. Pflugers Arch 466 (2014) 155–172.

[2] M. Galluccio, M. Tripicchio, L. Console, C. Indiveri, Bacterial over‑production of the functionally active human SLC38A2 (SNAT2) exploiting the mistic tag: a cheap and fast tool for testing ligands, Mol Biol Rep 51 (2024) 336-343.

[3] D. Yao, B. Mackenzie, H. Ming, H. Varoqui, H. Zhu, M.A. Hediger, J.D. Erickson, A novel system A isoform mediating Na+/ neutral amino acid cotransport. J Biol Chem 275 (2000) 22790-22797.

The proton translocation by many bioenergetic proteins involves the transfer of protons via a Grotthus-like mechanism through water and amino acid side chains. The likely protonation states and proton affinities (pKa) of amino acids involved in proton transfer are crucial for understanding the mechanisms of bioenergetic proteins. Two such bioenergetic proteins, respiratory complex I and multiple resistance and pH adaptation (Mrp) cation/proton antiporter, have been studied with high-resolution cryo-EM and MD simulations, which led to the identification of protein-bound functionally important water molecules [1, 2]. Recently, we showed the importance of correct charge state assignment in the conformational dynamics of protein and water molecules, as well as hydrogen bonding rearrangements [3]. Here, we applied conventional and recently developed state-of-the-art constant-pH simulation methods and tools [4, 5] to obtain detailed information on the likely protonation states of key amino acids of complex I and Mrp antiporter. We envisage data from our simulations will help in elucidating the fine-details of proton transfer pathways in these proteins.

[1] K. Parey, J. Lasham, D.J. Mills, A. Djurabekova, O. Haapanen, E.G. Yoga, H. Xie, W. Kühlbrandt, V. Sharma, J. Vonck, High-resolution structure and dynamics of mitochondrial complex I—Insights into the proton pumping mechanism, Science advances 7(46) (2021) eabj3221.

[2] Y. Lee, O. Haapanen, A. Altmeyer, W. Kühlbrandt, V. Sharma, V. Zickermann, Ion transfer mechanisms in Mrp-type antiporters from high resolution cryoEM and molecular dynamics simulations, Nature Communications 13(1) (2022) 6091.

[3] J. Lasham, A. Djurabekova, V. Zickermann, J. Vonck, V. Sharma, Role of Protonation States in the Stability of Molecular Dynamics Simulations of High-Resolution Membrane Protein Structures, The Journal of Physical Chemistry B (2024).

[4] N. Aho, P. Buslaev, A. Jansen, P. Bauer, G. Groenhof, B. Hess, Scalable Constant pH Molecular Dynamics in GROMACS, Journal of Chemical Theory and Computation 18(10) (2022) 6148-6160.

[5] P. Buslaev, N. Aho, A. Jansen, P. Bauer, B. Hess, G. Groenhof, Best Practices in Constant pH MD Simulations: Accuracy and Sampling, Journal of Chemical Theory and Computation 18(10) (2022) 6134-6147.

Mitochondria are well known as a hub of bioenergetic processes in cells, besides they also serve critical functions in cellular pathways, including life-death signalling, by maintaining homeostasis of calcium ions and reactive oxygen species (ROS). The inner mitochondrial membrane (IMM) is a barrier to the free flow of most ions. To facilitate their transport between the cytosol and the matrix, the IMM harbours a variety of transport proteins that regulate their passage, among these proteins are also mitochondrial potassium (mitoK) channels. The activation of mitoK channels is now a well-established phenomenon known for its cytoprotective effects which is particularly critical in tissues most sensitive to ischemia-reperfusion injury, such as the heart and the brain. In our research, we employed native guinea pig cardiomyocytes as a model system. The Western blot analysis showed the presence of the pore-forming subunit α of mitochondrial large conductance Ca²⁺-activated potassium (mitoBK_Ca) channel in purified mitochondria. The presence of mitoBK_Ca channels was also confirmed in electrophysiological experiments. Applying patch-clamp technique, we characterized mitoBK_Ca channel with a conductance of approximately 130 pS. The observed channel was voltage-dependent and Ca²⁺-sensitive - it exhibited the characteristic features of the mitoBK_Ca channel. Additionally, the mitoBK_Ca channel was blocked by paxilline, its well-known inhibitor. Moreover, we have shown that the mitoBK_Ca channel activity is redox-dependent. The oxidation of inner mitochondrial membrane environment by K₃[Fe(CN)₆] led to inhibition of the channel activity. Intriguingly, we found that illumination with a 820 nm infrared light could restore the mitoBK_Ca channel activity lost due to oxidation. The 820 nm wavelength corresponds to the absorption maximum of one of the copper centres (Cu_A) in cytochrome c oxidase (COX). We postulate that interplay between mitoBK_Ca channel and electron transport chain complexes could be one of molecular mechanisms underlying the protective effect of infrared radiation. Possibly, this interaction could be the core of future phototherapy preventing damages of ischemia-reperfusion injury.

Supported by the Polish National Science Centre (grants No. 2019/34/A/NZ1/00352 to AS).

Multidrug Resistance-Associated Protein 2 (MRP2/ABCC2) plays a pivotal role in the cellular efflux of organic anions, including drugs and bilirubin glucuronides, with its dysfunction leading to jaundice and Dubin-Johnson syndrome. Moreover, MRP2 has been identified as the determinant in anticancer therapy resistance, mediating efflux of many anticancer drugs such as paclitaxel and cisplatin. We present novel insights into the structural and functional modulation of MRP2, using cryo-EM to capture the architecture of rat Mrp2 in its autoinhibited state or bound to the drug probenecid. The autoinhibited conformation reveals a unique regulatory domain arrangement within the transmembrane domain cavity, restricting transporter activity. Phosphorylation of Ser922 and Ser926 triggers a significant increase in transport activity, as evidenced by in vitro phosphorylation studies, mass spectrometry, and functional assays in proteoliposomes following the fluorescent substrate 5(6)-Carboxy-2′,7′-dichlorofluorescein. The probenecid-bound structure uncovers two distinct drug-binding sites, providing a structural basis for the drug's modulatory effects on MRP2 activity. Experimental data, including ATPase activity assays and substrate transport analysis, underscore the importance of phosphorylation in modulating MRP2's function and the intricate relationship between drug binding and transporter regulation. This proposed work sheds light on the molecular dynamics of MRP2, offering potential avenues for therapeutic intervention in drug resistance and transport-related disorders. The findings underscore the critical role of structural modifications and kinase-mediated phosphorylation in determining MRP2's activity, laying the basis for future research into targeted drug development and treatment strategies for related pathological conditions.

[1] Mazza, T., Roumeliotis, T.I., Garitta, E. et al. Structural basis for the modulation of MRP2 activity by phosphorylation and drugs. Nat Commun 15, 1983 (2024). https://doi.org/10.1038/s41467-024-46392-8

To survive extreme alkalophilic and halophilic environments, bacteria and archaea have evolved specialized mechanisms for maintaining intracellular pH and facilitating sodium ion efflux. At the core of their adaptability, lies the crucial enzyme known as the Mrp (Multiple Resistance and pH adaptation) Na+/H+ antiporter, essential for survival and growth [1]. Its protein subunits responsible for proton translocation share sequence and structural similarities with the membrane domain of respiratory complex I, the first enzyme in the mitochondrial electron transport chain. Although high-resolution structural data on Mrp enzymes have recently become available, a comprehensive mechanistic understanding still remains elusive. In this study, we establish the fundamental basis for the proton translocation mechanism of Mrp antiporter. Our approach combines site-directed mutagenesis experiments and atomistic molecular dynamics (MD) simulations. We investigate various point mutations near a highly conserved histidine residue that resides at a junction of three putative proton transfer pathways. Changes in enzyme activity are correlated with the conformational changes at sidechain and backbone levels and hydration/dehydration effects, which are explored through long time scale wild-type and mutant MD simulations in various protonation states of critical titratable residues. Our findings lead to a model (histidine switch) of proton translocation in Mrp Na+/H+ antiporters [1,2], and expand our mechanistic understanding from bacterial adaptation in extreme environments to biological energy conversion by mitochondrial complex I [3,4].

[1] Y. Lee, O. Haapanen, A. Altmeyer, et al., Ion transfer mechanisms in Mrp-type antiporters from high-resolution cryoEM and molecular dynamics simulations, Nat Comm, 13, (2022) 6091.

[2] A. Djurabekova, J. Lasham, O. Zdorevskyi, V. Zickermann, V. Sharma, Long-range electron proton coupling in respiratory complex I — insights from molecular simulations of the quinone chamber and antiporter-like subunits, Biochem J, 481 2024 499–514.

[3] A. Djurabekova, O. Haapanen, V. Sharma, Proton motive function of the terminal antiporter-like subunit in respiratory complex I, BBA Bioenergetics, 1861(2020) 148185.

[4] M. Wikström, C. Pecorilla, V. Sharma, The mitochondrial respiratory chain, Enzymes, 54 (2023) 15-36.

The functional insertion, folding and assembly of proteins in biological membranes are essential for a variety of cell activity processes including cell signaling, transport of molecules in and out of the cell. Here we present an innovative approach using Surface-enhanced absorption spectroscopy (SEIRAS) to monitor translocon-unassisted membrane protein folding into artificial lipid bilayers (nanodiscs) during cell-free protein expression. For a first experiment nanodiscs were attached to a gold surface modified with Ni-NTA via a His-tag, while cell-free expression of the model protein bacteriorhodopsin (HsBR) was processing in the bulk solution. Time-resolved SEIRAS is solely sensitive to events taking place at the surface covered with the nanodiscs where the folding takes place and provides structural folding information. After the gene transcription and translation processes have been started, we could observe newly synthesized protein inserting into the nanodiscs and firstly forming secondary structures followed by tertiary structure indicating correct folding and integration of the chromohore all-trans retinal [1]. In a second round we investigated the microbial rhodopsins sensory rhodopsin I (HsSRI), sensory rhodopsin II (HsSRII) and channelrhodopsin II (CrChR2) in comparison to HsBR. These new microbial rhodopsins fail to properly bind retinal as indicated by the missing visible absorption in in-vitro cell expressions. SEIRAS experiments suggest that all investigated rhodopsins lead to the production of polypeptides and showed secondary structure formation. But the condensation of helices in tertiary structure formation was not accomplished properly indicating that the chromophore is not bound which seems to be mandatory for the correct folding process [2]. Our method is suitable to analyse folding processes and indicate steps for improvements in in-vitro expressions.

[1] A. Baumann, S. Kerruth, J. Fitter, G. Buldt, J. Heberle, R. Schlesinger, K. Ataka, In-Situ Observation of Membrane Protein Folding during Cell-Free Expression, PLoS One 11(3) (2016) e0151051.

[2] K. Ataka, A. Baumann, J.L. Chen, A. Redlich, J. Heberle, R. Schlesinger, Monitoring the Progression of Cell-Free Expression of Microbial Rhodopsins by Surface Enhanced IR Spectroscopy: Resolving a Branch Point for Successful/Unsuccessful Folding, Front Mol Biosci 9 (2022) 929285.

The emergence of antibiotic resistance has become an increasingly formidable global challenge since the turn of the 20^th century. Addressing this issue is of paramount importance, and compounds sourced from plants have emerged as among the most promising avenues for potential solutions. Unraveling the intricate mechanisms that underlie bacterial antibiotic resistance is a critical attempt that demands severe exploration. Therefore, our investigation examines deeply into studying the mechanism of action of menthol on antibiotic resistant Escherichia coli, a biologically active component which presents in essential oil of Mentha arvensis, in order to reveal potential therapeutic interventions. Through the application of gas chromatography and mass spectrometry, we have determined that menthol comprises a significant part, approximately 70%, of the essential oil's chemical composition.

Both essential oil and menthol have high antibacterial activity on kanamycin-resistant E. coli pARG-25 and the control strain E. coli BW25113, thus it is important to investigate the action mechanism of menthol. Particularly striking is menthol's profound impact on the kanamycin-resistant E. coli pARG-25 strain where it significantly reduces the total proton flux rate—a phenomenon conspicuously absent in the wild-type strain (E. coli BW 25113), where proton flux rates remain unaltered. Furthermore, menthol demonstrates 1.4-fold suppression of DCCD-sensitive ATPase activity in the kanamycin-resistant strain, contrasting with its negligible effect on the wild-type strain.

Menthol has considerable important inhibitory influence on the kanamycin-resistant E. coli pARG-25 strain, resulting in a notable twofold suppression of potassium flux rate, a crucial aspect of bacterial homeostasis. These multifaceted findings underscore menthol's broad-spectrum antimicrobial efficacy, which appears to be mediated through diverse mechanisms. These mechanisms include the modulation of bacterial membrane permeability, perturbation of ion flux—specifically proton and potassium—and modulation of DCCD-sensitive ATPase activity, suggesting a multi-pronged approach to antimicrobial action.

Taken together our investigation provides valuable insights into the antimicrobial properties of menthol and its potential as a therapeutic agent against antibiotic-resistant bacteria. These findings not only deepen our understanding of antibiotic resistance mechanisms but are also prospective for the development of novel treatment strategies to combat global health challenges.

cAMP receptor protein (CRP) is a transcriptional dual regulator that is involved in catabolite repression and functions differently based on the concentration of glucose, either up- or downregulating genes [1]. The impact of the CRP protein on proton flux (J_H+) in Escherichia coli in response to varying glucose concentrations was investigated.
E. coli BW25113 wild-type and Δcrp mutant were grown in peptone medium without glucose and with the addition of low (2 g L^-1) and high (8 g L^-1) glucose. The J_H+ was determined using a pH-selective electrode.
When cells were grown in media without glucose and low glucose was added in the assays, the total J_H+ in Δcrp was 32% lower than in the wild-type, but the flux conditioned by acids was not affected, and the difference was due to F_OF₁-ATPase. When high glucose was added in the assays, the total flux difference was not significant, but the flux conditioned by acids decreased by 39% and the input of F_OF₁-ATPase increased by 44%. In cells cultivated with low glucose, the addition of a low concentration of glucose in the assays resulted in a 27% decrease in total J_H+ compared to the wild-type. However, the contribution of F_OF₁-ATPase was not affected. In contrast, when a higher concentration of glucose was added in the assays, the J_H+ in the mutant strain decreased by 45%, but it was due to decreased F_OF₁-ATPase input.
When cells were grown with high glucose and low glucose was added in the assays, the total J_H+ did not differ, but the F_OF₁-ATPase input decreased by 32%.
Taken together, it can be concluded that when cells are grown without or with low glucose, CRP deletion decreases the H+ flux conditioned by acids. This decrease can be related to a lower glucose consumption rate. However, the impact of ATPase, varies depending on the external glucose concentration, thus most probably the regulation of other enzymes by CRP indirectly affects ATPase activity.

[1] B. Rieger, D.N. Shalaeva, A.C. Sohnel, W. Kohl, P. Duwe, A.Y. Mulkidjanian, K.B. Busch, Lifetime imaging of GFP at CoxVIIIa reports respiratory supercomplex assembly in live cells, Sci Rep 7 (2017) 46055.

Transport of adenine nucleotides (AdNs) across the inner mitochondrial membrane is central to cellular metabolism and is classically associated with the activity of adenine nucleotide translocases (ANTs), which perform the ATP/ADP exchange coupled with mitochondrial respiration. However, the regulation of the net content of AdNs in the matrix is accomplished by another group of carriers, the calcium-regulated mitochondrial ATP-Mg²⁺/P_i carriers (also named SCaMCs, del Arco and Satrústegui, 2004) which exchange ATP-Mg²⁺ or ADP for P_i. However, little is known about the physiological role of SCaMCs. Here, we have explored the role of the isoform SCaMC-1/SLC25A24, characteristic of proliferating cells exhibiting the Warburg effect. In contrast to other proposals (Maldonado et al., 2016), we show that ANTs, and not SCaMC-1, regulate the nucleotide exchange associated with respiration and the maintenance of the mitochondrial membrane potential in tumor and other proliferative cells.

To further explore the function of SCaMC-1, we focused on the idea that this carrier is activated by cytosolic calcium signals in the low micromolar range. So, we characterized the metabolic response of proliferating cells to calcium mobilization using agonists such as histamine or bradykinin in both WT and SCaMC-1-deficient proliferating cells. We observed that mitochondrial ATP levels increased upon calcium mobilization, but, in contrast with proposed ideas, independently of the stimulation of mitochondrial respiration. In fact, we show that calcium-dependent activation of SCaMC-1/SLC25A24 drives mitochondrial ATP import in response to agonists. Interestingly, this activity is specific for SCaMC-1, and cannot be rescued with other SCaMC isoforms. This novel mechanism is conserved in different proliferative cells and is responsible for the increases in mitochondrial ATP levels upon calcium mobilization. The physiological relevance of this process needs to be further studied.

del Arco A, Satrústegui J. (2004) Identification of a novel human subfamily of mitochondrial carriers with calcium-binding domains. J Biol Chem, 279:24701-24713.

Maldonado EN, DeHart DN, Patnaik J, Klatt SC, Gooz MB, Lemasters JJ. (2016) ATP/ADP Turnover and Import of Glycolytic ATP into Mitochondria in Cancer Cells Is Independent of the Adenine Nucleotide Translocator. J Biol Chem, 291:19642-19650.

The mitochondrial pyruvate carrier (MPC) is a mitochondrial inner membrane transporter, which links cytosolic and mitochondrial metabolism by transporting pyruvate into the matrix [1-2]. The functional human carrier is a heterodimer, formed of two homologous protomers [2]. It is ubiquitously expressed as MPC1/MPC2 in humans, with an additional dimer of MPC1L/MPC2 expressed in the testis [3]. Although the carrier is a putative drug target for diseases, such as diabetes, metabolic dysfunction-associated steatotic liver disease (MASLD), and neurodegeneration, little is known about its structure and transport mechanism.

In this work, a model of the human mitochondrial pyruvate carrier heterodimer was generated, based on predicted structures of the individual MPC1L and MPC2 protomers and known structural homologs of the transporter. By mutagenesis in the dimerisation interface and thermostability shift assays with a range of canonical MPC inhibitors, we have validated our structural model and identified residues essential for inhibitor binding in the functional heterodimer. Using radiolabelled pyruvate transport assays in proteoliposomes, we have also identified residues required for pyruvate transport, as well as key chemical features of MPC inhibitors required for high affinity transport inhibition. This information will be crucial for future investigations and for identifying medically relevant inhibitors of MPC.

[1] S. Tavoulari, C. Thangaratnarajah, V. Mavridou, M.E. Harbour, J. Martinou, E.R. Kunji, The yeast mitochondrial pyruvate carrier is a hetero‐dimer in its functional state, The EMBO Journal. 38 (2019). doi:10.15252/embj.2018100785.

[2] S. Tavoulari, T.J.J. Schirris, V. Mavridou, C. Thangaratnarajah, M.S. King, D.T.D. Jones, et al., Key features of inhibitor binding to the human mitochondrial pyruvate carrier hetero-dimer, Molecular Metabolism. 60 (2022) 101469. doi:10.1016/j.molmet.2022.101469.

[3] B. Vanderperre, K. Cermakova, J. Escoffier, M. Kaba, T. Bender, S. Nef, et al., MPC1-like is a placental mammal-specific mitochondrial pyruvate carrier subunit expressed in postmeiotic male germ cells, Journal of Biological Chemistry. 291 (2016) 16448–16461. doi:10.1074/jbc.m116.733840.

Uncoupling protein 1 (UCP1) from brown adipose tissue (BAT) carries out proton leak across the mitochondrial inner membrane to uncouple oxidative phosphorylation and release energy as heat, to help protect mammals from cold temperatures. The protein is activated by free fatty acids, which overcome inhibition by cytosolic purine nucleotides to induce proton leak activity. Different UCP1 isoforms (e.g. mouse, human, ovine) have been used in investigations on UCP1 in past scientific research, though whether or not these model proteins exhibit isoform specific differences in their ligand regulation and molecular properties is not clear. Targeting UCP1 for thermogenic energy expenditure is a potential therapeutic avenue to treat metabolic disease in humans, and so possible differences in these variants are important to identify.

Here, we have purified human, mouse, and ovine UCP1 recombinantly produced in yeast, and characterised the ligand interactions and activities of these isoforms in comparison to native ovine UCP1 (purified from lamb BAT). Ligand-induced thermostability shift analysis and liposome activity assays reveal protein unfolding profiles, nucleotide-induced stabilisation, and activation and inhibition behaviour in liposomes, indicative of intact protein for all isoforms, similar to native ovine UCP1. However, subtle differences in the relative thermostability observed in the presence of ligands suggest underlying bonding interactions may be different in particular isoforms.

Uncoupling protein 1 (UCP1), highly expressed in brown adipose tissue, plays a pivotal role in non-shivering thermogenesis by dissipating the mitochondrial proton motive force. When activated, UCP1 allows protons across the mitochondrial inner membrane bypassing ATP synthesis. Recent structures of UCP1 inhibited by purine nucleotides [1,2] reveal that these nucleotides block proton conductance by binding with high affinity to the central cavity of UCP1, preventing the necessary conformational changes [3]. This study investigates the nucleotide specificity of UCP1 inhibition. Using functional and structural analysis, we reveal how different nucleotide species have distinct affinities for UCP1. We also analysed the role of pH in the regulation of inhibition. Additionally, we explored how related mitochondrial carriers interact with nucleotides. UCP1 has evolved to bind nucleotides with high affinity, requiring only minimal changes to central cavity residues compared to related mitochondrial carriers. We also investigated which modifications in the central cavity have enabled the broad nucleotide specificity of UCP1. Our study suggests that UCP1 regulation may involve multiple nucleotides, contingent upon cellular conditions, highlighting the potential complexity of its regulatory mechanism. A complete understanding of the regulation of non-shivering thermogenesis is vital for harnessing the process to treat metabolic disease.

Uncoupling protein 1 (UCP1) is found in the inner mitochondrial membrane of brown adipocytes. In the presence of long-chain fatty acids (LCFA), UCP1 increases the H+ conductance to ‘short-circuit’ the proton-motive force, which, in turn, increases fatty acid oxidation and energy release as heat.

In the first part of my PhD project, I found the optimal conditions to produce UCP1 in yeast cells Saccharomyces cerevisiae and to purify the recombinant protein using mild detergents. Decreasing inducer amounts illustrates the need to tune the promoter for efficient solubilization of recombinant membrane proteins, not only in E. coli, where it is widely accepted but also in eukaryotic expression systems. Our protocol removes toxicity of the expression, is cost-effective, suitable for large-scale culture volume, and consequently increases final protein purification yield even in minimal media. The proposed strategy is being extended to other classes of membrane proteins (Fine-tuning of the GAL10 promoter and growth conditions for recombinant production of the mitochondrial uncoupling protein UCP1 in yeast saccharomyces cerevisiae. In preparation).

Secondly, my lab’s recent findings about UCP1’s inhibition using molecular dynamics[1] that fit with the new experimental ones obtained by cryo-EM[2] led to the discovery of an interesting mutant that could have therapeutic potential in fighting obesity and diabetes and help to understand the mechanism of action of UCP1. We previously showed by respiration experiments using a high-resolution O2k-respirometer on yeast spheroplasts that the UCP1(F88I187W281AAA) triplet mutation is highly activated by free fatty acids even in the presence of inhibitory nucleotides. The strong uncoupling activity of this mutant is still unresolved despite the published structures. Therefore, we are combining ligand binding measurements by fluorescence on purified proteins with proton conductance after reconstitution into liposomes to establish the molecular mechanisms of UCP1 regulation.

[1] A. Gagelin, C. Largeau, S. Masscheleyn, M.S. Piel, D. Calderón-Mora, F. Bouillaud, J. Hénin, B. Miroux, Molecular determinants of inhibition of UCP1-mediated respiratory uncoupling, Nat Commun 14 (2023).

[2] S.A. Jones, P. Gogoi, J.J. Ruprecht, M.S. King, Y. Lee, T. Zögg, E. Pardon, D. Chand, S. Steimle, D.M. Copeman, C.A. Cotrim, J. Steyaert, P.G. Crichton, V. Moiseenkova-Bell, E.R.S. Kunji, Structural basis of purine nucleotide inhibition of human uncoupling protein 1, Sci Adv 9 (2023).

Riboflavin (Rf) transporter deficiency 2 (RTD2, OMIM #614707) is a rare human neurological disorder caused by mutations in the SLC52A2 gene, encoding the Rf transporter 2 (RFVT2). Clinical manifestations include sensorineural hearing loss, bulbar dysfunction, respiratory compromise, and muscle weakness; symptomatology could be ameliorated by Rf treatment. The biochemical role of this vitamin is linked to its conversion into the redox cofactors FMN and FAD, crucial for mitochondrial bioenergetics and, therefore, for neurons [1].

To address the structure-function relationship of RFVT2, WT and mutants were overexpressed in E. coli, and the purified proteins were reconstituted in proteoliposomes: all the mutants were active, even if performing a lower affinity for Rf [2].

Based on recently built homology models RFVT2, dimerization was predicted [2,3]. The final demonstration was obtained using the GPCA assay. Preliminary results also suggest that some variants have a reduced ability to dimerize.

To investigate the effect of RFVT2 mutations on cellular economy, we used both patients’ fibroblasts and iPSC-derived motor neurons. The increase in the level of specific chaperones and the altered Ca²⁺-mediated response clearly demonstrated that cells carrying altered RFVT2 suffer from profound proteotoxic stress, probably due to an impairment of the correct maturation of RFVT2 rather than a Rf deficiency. The molecular rationale of Rf therapy needs further investigation.

[1] M.Tolomeo, A. Nisco, P. Leone, M. Barile, Development of Novel Experimental Models to Study Flavoproteome Alterations in Human Neuromuscular Diseases: The Effect of Rf Therapy, Int J Mol Sci, 21 (2020) 5310.

[2] L. Console, M. Tolomeo, J. Cosco, K. Massey, M. Barile, C. Indiveri, Impact of natural mutations on the riboflavin transporter 2 and their relevance to human riboflavin transporter deficiency 2, IUBMB Life, 74 (2022) 618-628.

[3] O. B. Mariem, S. Saporiti, U. Guerrini, T. Laurenzi, L. Palazzolo, C. Indiveri, M. Barile, E. De Fabiani, I. Eberini, In silico investigation on structure-function relationship of members belonging to the human SLC52 transporter family, Proteins, 91 (2023) 619-633.

VDACs are Outer Membrane mitochondrial proteins, allowing cross-talk between the organelle and cytosol and the transport of anions, cations, ATP, Ca²⁺ and metabolites. VDAC is a critical player in mitochondria mediated apoptosis. In addition, VDAC1 is now considered a general hub on the surface of mitochondria, and interacts with over 154 proteins.

Most pathological conditions lead to dysfunction of the mitochondria. Striking evidence exist of the generalized overexpression of VDAC1 at the onset of NCDs like tumors and cardiovascular diseases, and with age. This indicates that VDAC1 overexpression is a hallmark of disease (2). VDAC1 gene can thus be seen as a signaling hub where the overexpression of the protein is decided.

In this work we focus our attention on the mechanisms impacting on the gene-expression regulation of VDAC1.

Regulatory signatures of VDAC1 gene were validated experimentally. Molecular pathways involved in controlling cell growth, proliferation, differentiation, apoptosis and bioenergetics metabolism characterize VDAC1 gene regulation. VDAC1 transcription is increased when cells are exposed to glutamine and serum depletion or controlled hypoxia. Moreover, the methylation profile of VDAC1 genes promoter region suggest a mechanism of gene expression control inhibiting transcription factors activation. We also identified the regulatory pathways regulating over-expression of VDAC1 gene during apoptosis stimulated by anti-cancer drugs like cisplatin.

These data show that VDAC1 has a crucial role in diseases since it is involved in cell processes at the beginning of pathological cascade.

1) De Pinto V. Biomolecules (2021) 11:107.

2) Shoshan-Barmatz V, Shteinfer-Kuzmine A, Verma A. Biomolecules (2020) 10: 1485.

Ackn: PNRR M4C2­Investment 1.4­ CN000041, PRIN 2022NLLTRJ

The structural knowledge of complex I (NADH:ubiquinone oxidoreductase) has significantly improved in recent years due to advances in cryo-EM techniques [1]. Human complex I has 45 subunits and more than 20 assembly factors have been described. While the pathways of complex I assembly are known in considerable detail [2], the structures of assembly intermediates, assembly factors and their individual functions have remained largely unclear.

More insights into the assembly process from a structural perspective have been provided by our recent efforts focusing on the assembly factor NDUFAF1 and its associated assembly intermediates [3]. Assembly intermediates of the early and late P_P module demonstrate that the subunits ND2 and NDUFC2, in conjunction with the assembly factors NDUFAF1 and CIA84 provide a nucleation point for module assembly.

In order to better understand the assembly process and to provide additional information regarding the assembly, we are now investigating the role of the subsequent assembly factors in complex I assembly.

[1] K. Parey, C. Wirth, J. Vonck, V. Zickermann, Respiratory complex I - structure, mechanism and evolution, Curr. Opin. Struct. Biol., 63 (2020) 1-9.

[2] S. Guerrero-Castillo, F. Baertling, D. Kownatzki, H.J. Wessels, S. Arnold, U. Brandt, L. Nijtmans, The assembly pathway of mitochondrial respiratory chain complex I. Cell Metab., 25(1) (2017) 128-139.

[3] J. Schiller, E. Laube, I. Wittig, W. Kühlbrandt, J. Vonck, V. Zickermann, Insights into complex I assembly: Function of NDUFAF1 and a link with cardiolipin remodeling, Science Advances, 8 (2022) eadd3855.

ATP is a key signalling molecule in pancreatic β-cells as its increase causes Katp-sensitive potassium channels to close, dissipating the plasma membrane potential and triggering insulin secretion via further steps. Understanding the regulation of mitochondrial ATP synthase, the main source of ATP in β-cells, is thus crucial and can potentially be used in designing new therapeutic agents for Type 2 Diabetes Mellitus (T2DM). The best-known endogenous regulator of ATP synthase is ATPase inhibitory factor 1 (ATPIF1). The mechanistic way this protein works is still a subject of discussion. Our studies in INS1E model cells have shown that ATPIF1 is important in coupling the amount of available substrate and adequate ATP production [1,2]. In this study, we used pancreatic islets isolated from ATPIF1 knock-out (KO) mice and investigated whether the absence of ATPIF1 will impact the architecture of the mitochondrial network and the overall morphology of pancreatic islets. We employed Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) and high-resolution Structured Illumination Microscopy (SIM) to monitor the changes. The obtained microscopy data were used to segment mitochondria and insulin granules and were further processed by computational analysis. Moreover, insulin and glucagon expression was also studied by immunohistochemistry and compared between pancreatic islets of ATPIF1 KO and wild-type (WT) mice while also considering their age and gender. Altogether, the obtained data provide further insight into the emerging role of ATPIF1 in the physiology of pancreatic β-cells.

A grant from the Czech Scientific Foundation 22-02203S supported this project.

[1] A. Kahancová, F. Sklenář, P. Ježek, A. Dlasková, Overexpression of native IF1 downregulates glucose-stimulated insulin secretion by pancreatic INS-1E cells, Sci. Reports 2020 101 10 (2020) 1–13. https://doi.org/10.1038/s41598-020-58411-x.

[2] A. Kahancová, F. Sklenář, P. Ježek, A. Dlasková, Regulation of glucose-stimulated insulin secretion by ATPase Inhibitory Factor 1 (IF1), FEBS Lett. 592 (2018) 999–1009. https://doi.org/10.1002/1873-3468.12991.

Mitochondria are important cellular hubs for the integration of metabolic and signalling pathways, capable of communicating with other organelles through dynamic membrane contact sites. Recently, we showed that mitochondria establish contact sites with the nucleus [1], termed ‘nucleus-associated mitochondria’ (NAM). We also demonstrated that their formation is promoted in response to stress through the involvement of the mitochondrial translocator protein (TSPO) [1]. To expand the knowledge around the functional significance of NAM, we assayed their role in Ca²⁺ homeostasis. For this purpose, we employed neuronal cells and focused on nuclear Ca²⁺ dynamics ([Ca²⁺]_n), which are vital for coupling electrical activity with chromatin organisation and gene expression in this cell type.

By using human neuroblastoma SH-SY5Y cells, high-resolution imaging techniques and proximity labelling, we found that TSPO interacts with the chromatin- and lamin-binding protein thymopoietin (TMPO/LAP2), and that the interplay between the two proteins is a key factor for mito-nuclear interaction and communication. Impaired activity in both proteins reduces the frequency of NAM and impairs nuclear Ca²⁺ uptake, leading to loss of homeostatic cell signalling and alterations in gene expression. Whole transcriptome sequencing confirmed differences in Ca²⁺-regulated genes following NAM disruption, as well as alterations in Ca²⁺ channels on the nuclear envelope, including inositol-1,4,5-trisphosphate (IP₃) and ryanodine (RyR) receptors.

Our data advance the molecular understanding of the NAM-regulating machinery, highlighting for the first time the involvement of chromatin- and lamina-remodelling elements such as TMPO/LAP2. Therefore, we propose the existence of a molecular axis linking NAM to genomic responses through Ca²⁺-mediated signal transduction.

Impaired mitochondrial bioenergetics and defective endo/sarcoplasmic reticulum (ER/SR) function have been individually reported in skeletal muscle under degenerative conditions in both humans and rodents. However, the dynamic interplay between these two key organelles under physiological conditions as well as its contribution to skeletal muscle degeneration remain elusive. Here, we characterize the contribution of mitochondria-ER/SR communication to skeletal muscle biology and function in wildtype and mitofusin 1 and/or 2 knockdown mice. Experiments were carried out with male mice aged 10-12 weeks in a temperature-controlled room on a 12-h light/dark cycle. Mfn1/2^f/f (control), Mfn1^f/f/HSA^Cre, Mfn2^f/f/HSA^Cre and Mfn1/2^f/f/HSA^Cre mice were treated for 2 weeks with Doxycycline (1 mg/mL) in 5% sucrose-supplemented drinking water for deletion of mitofusin 1 and/or 2 in the skeletal muscle, followed by 4 weeks of washout. Our findings demonstrate that combined skeletal muscle deletion of mitofusins 1 and 2, critical proteins involved in mitochondria-ER tethering, is sufficient to induce a severe reduction in skeletal muscle mass, contractility properties and running distance (~50% decrease) when compared with control littermates. In addition, conditional deletion of mitofusin 1 and 2 modifies mitochondrial and SR morphology, impairs Ca²⁺ release units and induces changes in the levels of proteins involved in ER/SR calcium handling and ER stress in skeletal muscle. It is important to highlight that these transgenic mice do not display dysfunctional skeletal muscle mitochondrial bioenergetics in single fiber analysis. Of interest, mfn1/2 KO mice display impaired neuromuscular junction and fiber type switching to a oxidative metabolism. The presence of a single mitofusin (either Mfn1 or Mfn2) is sufficient to maintain those ER/SR changes described above. Together, these results place mitofusin 1 and 2 as critical players in (patho)physiological changes in skeletal muscle. Finnaly, unraveiling the molecular mechanisms of mitochondrial-ER/SR signaling and regulation will provide insights into the most fundamental cellular adaptive processes and perhaps uncover new druggable targes that will open up new strategies to treat skeletal muscle disease.

Pancreatic β-cells play an important role in the regulation of energy metabolism by secretion of the hormone insulin, and their dysfunction is one of the causes of type II diabetes mellitus. A crucial step in the insulin-secreting signaling pathway is an increase in respiratory chain activity in the mitochondria, which occurs after an increase in blood glucose concentration. Mitochondrial morphology is a key factor regulating respiratory chain activity since changes in cristae are linked to the conformation of respiratory chain supercomplexes and to efficiency of protonic coupling between FoF1 ATP-synthase and the respiratory chain [1]. Here, one of the main actors is OPA1, a GTPase of the dynamin protein family, which mediates the fusion of the inner mitochondrial membrane and regulates the shape of mitochondrial cristae. Its best-studied regulatory mechanism is specific proteolytic cleavage, mediated, among others, by OMA1, an integral membrane protease activated by stress stimuli [2]. What role this protease plays in the regulation of mitochondrial activity in β-cells is the subject of our study. We examined its expression and activity in model pancreatic beta cells, INS1E, under different conditions, such as different substrate levels or elevated reactive oxygen species levels. Moreover, to directly address its impact on INS1E cells, we performed OMA1 silencing. Subsequent analysis of mitochondrial network by superresolution and electron microscopy revealed altered mitochondrial network distribution, and changes in cristae width. As expected, mitochondrial morphology changes were accompanied by altered bioenergetics profile of these cells.

Grant 22-02203S from the Czech Scientific Foundation supported this project.

[1] P. Ježek, A. Dlasková, Dynamic of mitochondrial network, cristae, and mitochonnucleoids in pankreatické β-cells, (2019). https://doi.org/10.1016/j.mito.2019.06.007.

[2] R. Anand, T. Wai, M.J. Baker, N. Kladt, A.C. Schauss, E. Rugarli, T. Langer, The i-AAA protease YME1L and OMA1 cleave OPA1 to balance mitochondrial fusion and fission, Journal of Cell Biology 204 (2014) 919–929. https://doi.org/10.1083/jcb.201308006.