Conference Agenda

NADH:ubiquinol oxidoreductase or Complex I is a key enzyme of the respiratory chains. It catalyses proton coupled-electron transfer (PCET) reactions that contribute to the creation of a proton motive force across the membrane, powering ATP production via ATP synthase ^[1,2]. Despite decades of biochemical, biophysical, and structural studies, the PCET mechanism of Complex I remains unclear and highly debated ^[3,4]. By integrating biophysical proton transport assays in proteoliposomes with multi-scale simulations we engineer, isolate, and study here how the antiporter modules of the bacterial Complex I catalyse the proton transport across the membrane. Our findings illustrate key proton pathways in the membrane domain of Complex I, and how functional elements modulate the rate of proton conduction. Our combined findings provide a blueprint for understanding the long-range proton translocation mechanism in Complex I.

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

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

[3] Kaila V. R. I. Accounts of Chemical Research 54,(2021), 4462-4473

[4] Kravchuk, V. et al. “A universal coupling mechanism of respiratory complex I.” Nature vol. 609,7928 (2022): 808-814.

[5] Beghiah A et al. (in review)

Potential differences for electron transfer through biopolymers or in bio-nano setups can amount to several 100 mV. We describe these situations by Pauli-Master equations that are based on Marcus’ theory of charge transfer between self-trapped electrons and that obey Kirchhoff's current law. We present analytical and numerical current-potential curves and electron populations for multi-site model systems and biological electron transfer chains. Based on these, we provide empirical rules for electron populations and chemical potentials along the chain. The Pauli-Master mean-field results are validated by kinetic Monte Carlo simulations. We discuss the biochemical and evolutionary aspects of our findings [1]. In the vital respiratory complex I, electron transfer is followed by protein-quinone unbinding, which in turn triggers proton dislocation against a pH gradient. We estimate the entropic contributions to this process in lipid bilayers and obtain an entropy drive of up to 50 kJ/mol. We suggest an entropic zipper model and discuss it in the context of the bioenergetics and the structure of complex I [2].

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

[2] J. Haxhija, F. Guischard, T. Koslowski, A trick of the tail: computing the entropic contribution to the energetics of quinone-protein unbinding, PCCP 25 (2023) 27498-27505

The respiratory Complex I constitutes the initial entry point of electrons in the respiratory chain. This highly intricate redox-driven proton pump catalyzes the electron transfer from NADH to quinone (Q), coupled to the translocation of protons across the membrane domain, 200 Å away from the catalytic site. Despite recent advances, the molecular principles of this long-range proton-coupled electron transfer (PCET) process remain one of the main unresolved questions in the field of bioenergetics [1,2]. We recently identified conserved loop elements and transmembrane helices around the Q-binding site that undergo conformational changes during the Q reduction and could initiate the proton pumping in the membrane subunits [3]. Here, we combine multiscale simulations with biochemical and biophysical experiments, to probe how the redox signal is transduced to the membrane domain by evolutionary conserved functional coupling elements. We propose a switching network that mediates the coupling between the redox process and the proton pumping, with key implications in the long-range coupling mechanism of Complex I.

[1]. V.R.I. Kaila, Long-Range Proton-Coupled Electron Transfer in Biological Energy Conversion: Towards Mechanistic Understanding of Respiratory Complex I. J. R. Soc. Interfaces 15 (2018). 20170916.

[2]. V.R.I. 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.

[3]. H. Kim, P. Saura, M.C. Pöverlein, A.P. Gamiz-Hernandez, V.R.I. Kaila, Quinone Catalysis Modulates Proton Transfer Reactions in the Membrane Domain of Respiratory Complex I. J. Am. Chem. Soc. 145 (2023) 17075-17086.

When looking at the emergence of life as a common denominator of current living, one feature seems shared among most living organisms : the use of bioenergetic electron transport chains to produce energy through chemiosmosis. At the origin of life, the latter system where metalloenzymes couple transmembrane redox and cation gradients to power phosphate condensation could have emerged in hydrothermal vent chimneys walls bearing both electrochemical and proton gradients thanks to the presence of inorganic redox catalysts (i.e. minerals) powering a primitive bioenergetic-like system [1].

To simulate this system, we focused on the unexplored insertion inside liposomes membranes of the redox mineral « green rust » [2], considered as an inorganic precursor of current bioenergetic proteins, due to their structural similarities [1].

We developped a nano-inorganic system analogous to current bioenergetic proteins, both similar in terms of size and hydrophobicity [2], and harvesting redox-reactivity [3] and channel-like interlayer-spaces [1]. The use of hydrophobic amino-acids as coating agent brings our mineral system closer to current metallo-enzymes, feeding the inorganic-to-organic transition in bioenergetic systems.

The incorporation of functionnalized green rusts within liposomes membranes allows to test their ability to perform the chemiosomotic reactions performed by current bioenergetic metallo-enzymes, thus being relevant candidates for inorganic chemiosmosis at the emergence of life.

[1] S. Duval, F. Baymann, B. Schoepp-Cothenet, F. Trolard, G. Bourrié, O. Grauby, E. Branscomb, M.J. Russell, W. Nitschke, Fougerite: the not so simple progenitor of the first cells, Interface Focus 9 (2019) 20190063. https://doi.org/10.1098/rsfs.2019.0063.

[2] N. Gaudu, O. Farr, G. Ona-Nguema, S. Duval, Dissolved metal ions and mineral-liposome hybrid systems: Underlying interactions, synthesis, and characterization, Biochimie 215 (2023) 100–112. https://doi.org/10.1016/j.biochi.2023.09.009.

[3] O. Farr, N. Gaudu, G. Danger, M.J. Russell, D. Ferry, W. Nitschke, S. Duval, Methanol on the rocks: green rust transformation promotes the oxidation of methane, Journal of The Royal Society Interface 20 (2023) 20230386. https://doi.org/10.1098/rsif.2023.0386.

Despite the vast diversity of life, a surprisingly small group of core mmachineries is responsible for the myriad microbial bioenergetic solutions. Enzymes that contain transition metals, iron-sulfur centers, flavins, hemes, or other prosthetic groups catalyze the majority of bioenergetically relevant reactions. These cofactors can serve as electron transfer sites or play crucial roles in catalysis. Many of these complexes can directly donate (or receive) electrons to other proteins, or instead, transfer them to coenzymes like NADH or quinones. The widespread use of cofactors in biological processes, their universal conservation, and their proven catalytic potential make them essential for any living system and underscores the remarkable efficiency and adaptability of biological systems.Therefore, investigating their evolution can shed light on which metabolic pathways were possible over time and which were not.

Here we present our recent findings in terms of the evolution of the biosynthesis of several cofactors and integrate the results in terms of the evolution of bioenergetic systems. Understanding their evolution not only deepens our knowledge of life's evolution but also reveals the ingenious strategies that have allowed organisms to thrive in diverse environments.

At the thermodynamic limit of life, many anaerobic bacteria and archaea rely on the membrane-bound Rnf complex to power cellular metabolism [1]. This central respiratory enzyme of many pathogens catalyses the electron transfer from ferredoxin (E₀’ = -500 mV) to NAD⁺ (E₀’ = -320 mV), coupling the process to the translocation of sodium ions (Na⁺) across a bacterial membrane, and powering the synthesis of ATP. To probe the molecular mechanism of the elusive redox-driven Na⁺-translocation in Rnf, we integrate here cryo-electron microscopy (cryo-EM) and large-scale atomistic molecular simulations with biochemical functional assays. We identify key conformational changes in the membrane domain of the complex, as well as conserved residues involved in Na⁺-binding and translocation. Based on our combined findings, we suggest that Rnf operates via an alternating access mechanism, where the conformational transition is driven by the redox-triggered Na⁺-binding. Our study provides valuable insights into the molecular mechanism of the redox-driven Na⁺-pumping in the Rnf machinery.

[1] E. Biegel, S. Schmidt, J. M. González, V. Müller, Biochemistry, evolution and physiological function of the Rnf complex, a novel ion-motive electron transport complex in prokaryotes., Cell. Mol. Life Sci., 68 (2011) 613–634.

[2] A. Kumar, J. Roth, H. Kim, P. Saura, S. Bohn, T. Reif-Trauttmansdorff, A. Schubert, V. R. I. Kaila, J. M. Schuller, V. Müller, Molecular principles of redox-coupled sodium pumping of the ancient Rnf machinery., (In review).

Wolbachia pipientis is a Rickettsial bacterium that infects a wide variety of insects, including mosquitoes, fruitflies, and butterflies. It is transmitted maternally. In mutualistic associations, Wolbachia can provide the host with metabolites or assist in maintaining the chemical homeostasis of the host cell [1]. The main species of Wolbachia that infect Drosophila spp. are WbPop (Drosophila simulans) and WbMel (Drosophila melanogaster) [2]. In the fly, WbPop has higher bacterial density, cytoplasmic incompatibility, vertical transmission, and invasion capacity than WbMel. Additionally, it significantly enhances fertility and causes cytological defects. These differences can impact the interaction between Wolbachia and Drosophila, potentially leading to bioenergetic changes in the host [3]. Preliminary data indicate that oxygen consumption is enhanced in infected Drosophila as compared to controls. The mechanism for this enhancement is under study.

[1] Bishop, C. and Asgari, S.. Altered gene expression profile of Wolbachia pipientis wAlbB strain following transinfection from its native host Aedes albopictus to Aedes aegypti cells. Molecular Microbiology, 115(2021), 1229-1243.

[2] Bubnell, J. E., Fernandez-Begne, P., Ulbing, C. K. S. and Aquadro, C. F.. Diverse wMel variants of Wolbachia pipientis differentially rescue fertility and cytological defects of the bag of marbles partial loss of function mutation in Drosophila melanogaster. G3, 11 (2021) 1-12.

[2] Zhukova, M. V. and Kiseleva, E. The virulent Wolbachia strain wMelPop increases the frequency of apoptosis in the female germline cells of Drosophila melanogaster. BMC Microbiol, 12(2012) 1-12.

Autotrophic theories for the origin of metabolism posit that the first cells satisfied their carbon needs from CO2 and were chemolithoautotrophs that obtained their energy and electrons from H2. The acetyl-CoA pathway of CO2 fixation is central to that view because of its antiquity: Among known CO2 fixing pathways it is the only one that is i) exergonic, ii) occurs in both bacteria and archaea, and iii) can be functionally replaced in full by single transition metal catalysts in vitro. In order to operate in cells at a pH close to 7, however, the acetyl-CoA pathway requires complex multi-enzyme systems capable of flavin-based electron bifurcation that reduce low potential ferredoxin—the physiological donor of electrons in the acetyl-CoA pathway—with electrons from H2. How can the acetyl-CoA pathway be primordial if it requires flavin-based electron bifurcation? We have shown that native iron (Fe0), but not Ni0, Co0, Mo0, NiFe, Ni2Fe, Ni3Fe, or Fe3O4, promotes the H2-dependent reduction of aqueous Clostridium pas- teurianum ferredoxin at pH 8.5 or higher within a few hours at 40 °C, providing the physiological function of flavin-based electron bifurcation, but without the help of enzymes or organic redox cofactors [1]. H2-dependent ferredoxin reduction by iron ties primordial ferredoxin reduction and early metabolic evolution to a chemical process in the Earth’s crust promoted by solid-state iron, a metal that is still deposited in serpentinizing hydrothermal vents today.

As a part of the respiratory chain, Cytochrome c Oxidase catalyzes the reduction of the final electron acceptor of aerobic respiration, namely molecular oxygen. The free energy available in this reduction is utilized by the enzyme in order to pump proton across the energy-transducing membrane, against the electrochemical gradient, and by doing so manages to utilize even more of the energy available. This enzyme has been studied for a long time, and while a great wealth of information is known today, some questions remain. These are mainly with regards to how the enzyme prevents back-leakage of protons to the thermodynamically favourable side of the membrane, or to the arguably most favoured destination; the protonation of the reduced oxygen species. One promising technique for the acquisition of this information is time-resolved serial femtosecond X-ray crystallography, in which a slurry of microcrystals can be illuminated by a pump-laser, inititing the event under investigation, awhich is followed by X-rays probing the structure. Unlike proteins with a chromophore that could be used for the reaction initiation, there are several hurdles to making this work in the crystalline form of the enzyme. I will here present the approach of using a photolabile oxygen cage, the obstacles encountered as well as how to bypass them. The novel structural signals of the bacterial ba₃-type oxidase which can be seen from the time-resolved oxygenation of the enzyme are centered around water molecules just above as well as below of the active site, and have significant implications for the mechanism of the enzyme.

Additionally, I will present results from the other method of choice in my PhD; quantum chemical calculations. I will here present novel insights into the cytochrome c oxidase mechanism obtained from high level multiconfigurational ab initio calculations of the active site.

The individual reaction steps in the proton pumping cycle of the bc₁ complex are well known but the mechanisms which suppress the thermodynamically more favorable short-circuit reactions are enigmatic. The bc₁ complex would not pump protons without suppression of these short-circuit reactions.

Here we develop an in-silico model of the bc₁ complex and analyze free energy profiles of bifurcation and short-circuit pathways. The model assumes that the UQ intermediates H-bond to His161 on the ISP which accepts the first proton. We identified 3 short circuit reactions pathways: slip where both electrons are passed to Cytc, and type I and type II leak where electrons are passed from b_L to FeS via UQ or UQH₂, respectively, at Q_o. Simulations of the original model showed electron flux was dominated by the short-circuit reactions. It was then adapted by changing the binding energies between the UQ intermediates at the Q_o site and His161 to give a final model that had negligible slip and leak, even when inhibited with antimycin.

Applying the framework of transition-state theory, we found that high energy states in the free energy profile suppress flux through the following step. Slip was suppressed because breaking the H-bond between UQS and the ISP on its movement to the c₁ conformation raises the energy of the state so that electron transfer (eT) from FeS to c₁ is suppressed. Type-I leak was suppressed because the oxygen facing His161 of the single-protonated UQS has an unpaired electron and so forms a very weak H-bond. This raises the state’s energy and eT from b_L to UQS occurs at low rates. In contrast, the subsequent reaction in the bifurcation pathway is the release of the second proton to its hydrogen bonded partner, which is much faster. Type II leak was suppressed by assuming that unprotonated UQS only forms a strong H-bond to His161 when FeS is reduced, similar to stigmatellin. This slows the eT from b_L to UQS but not from UQS to b_L during bifurcation because FeS is reduced at this time.

In this way, the structure and substrate-binding of the Qo site results in free energy profiles that naturally suppress the short circuit reactions while allowing high rates of bifurcation. We propose that this is the mechanism of suppression and hence the mechanism of proton pumping in the bc₁ complex.

[FeFe]-hydrogenase from Thermotoga maritima is composed of three subunits harbourig a total of 10 FeS clusters, a FMN and an H-cluster [1]. It shares its modular architecture with an entire family of flavin containing iron-sulfur proteins [2]. Some of the FeS clusters are localized in mobile domains. The enzyme catalyzes the reduction of protons from NADH and reduced ferredoxin during glucose fermentation and can also reduce NAD+ and Fd with electrons from H₂ in vitro. Both, NADH oxidation and electron confurcation from NADH and ferredoxin are supposed to occur on the same flavin. As for other flavoenzymes featuring a single flavin and reacting with NAD(P)H, ferredoxin and a further two-electron substrate their reaction mechanism defies our current understanding of flavin-based electron bi/confurcation.

We studied the redox and spectral properties of the flavin and the iron-sulfur centers in HydABC from Thermotoga maritima on the apo-protein and the isolated subunits. The redox potentials of the FeS clusters thereby covered a broad range from -270 mV to below -500 mV. Redox- and magnetic interactions among the iron-sulfur centers and between the iron-sulfur centers and the flavin were detected. The redox properties of the flavin varied between the apo-enzyme and the isolated HydB subunit and were influenced by the presence of the substrate NADH. These results lay a basis to further investigate the mechanism of electron bifurcation in HydABC and related enzymes.

[1] Furlan C, Chongdar N, Gupta P, Lubitz W, Ogata H, Blaza JN, Birrell JA (2022) Structural insight on the mechanism of an electron-bifurcating [FeFe] hydrogenase, eLife 11, e79361.

[2] Zuchan K, Baymann F, Baffert C, Brugna M, Nitschke W (2021) The dyad of the Y-junction and a flavin module unites diverse redox enzymes. Biochim Biophys Acta 1862, 148401.

Photosynthesis is highly sensitive to the lumenal pH. When protons accumulate in the lumen, the oxidation of plastoquinol (PQH₂) in the cytochrome (cyt) b₆f complex is slowed, representing a protective mechanism, termed photosynthetic control. In the cyt b₆f complex, the oxidation of PQH₂ is coupled with PQH₂ deprotonation. The His128 residue of iron-sulfur protein accepts the first proton from PQH₂. When [2Fe-2S] cluster donates an electron to cyt f, the proton is released to the lumen via a channel lined by polar uncharged residues. The Glu78 residue of subunit IV presumably accepts the second proton from plastosemiquinone followed proton diffusion into the lumen via a putative channel lined by polar charged residues of subunit IV, cyt b₆, and PetG. The role of His128 in the photosynthetic control is well known. However, elucidating the role of Glu78 in the sensitivity of PQH₂ oxidation to lumenal pH has received little attention.

We compare two competitive inhibitors of PQH₂ oxidation, 2,4-dinitrophenyl ether of 2-iodo-4-nitrothymol (DNP-INT) and 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB), in terms of their sensitivity to lumenal pH. Using isolated thylakoids of higher plants (Pisum sativum, Arabidopsis thaliana), we demonstrated that low pH enhanced the inhibitory activity of DNP-INT but limited the inhibitory activity of DBMIB. The latter was substantially retarded in the pgr1 arabidopsis mutant, in which the pKa of His128 is increased, implying that the sensitivity of DBMIB to lumenal pH relates to protonation of His128. In contrast, the inhibitory activity of DNP-INT in pgr1 mutant was slightly higher than that in wild type. We further applied valinomycin, which in complex with K⁺ (valinomycin:K⁺) is able to shield the negative charge of Asp and Glu residues, so it was proposed that valinomycin:K⁺ blocks proton transfer via the channel starting only from Glu78. Indeed, valinomycin enhanced the inhibitory activity of DNP-INT whereas it did not affect that of DBMIB.

The opposite sensitivity of DNP-INT and DBMIB to lumenal pH opens a new perspective for their use to investigate the molecular mechanisms of proton fluxes in the cyt b₆f complex.

This work was supported by Russian Science Foundation №23-14-00396 (https://rscf.ru/project/23-14-00396/).

Marine Diatoms contribute to oxygenic photosynthesis and carbon fixation and handle large changes under variable light intensity on a regular basis. The unique light-harvesting apparatus of diatoms are the Fucoxanthin-Chlorophyll a/c-binding proteins (FCPs). Here, we show the enhancement of Chlorophyll a/c (Chl a/c), Fucoxanthin (Fx), and Diadinoxanthin (Dd) marker bands in the Raman spectra of the diatoms Fragilariopsis sp and P. tricornutum , which allows distinction of the pigment content in the cells grown under low-(LL) and high-light (HL) intensity at room temperature. Reversible LL-HL dependent conformations of Chl c, characteristic of a planar and nonplanar configuration of the porphyrin macrocycle, and the presence of five-and six-coordinated Chl a/c with weak axial ligands are observed in the Raman data. Under HL the energy transfer from Chl c to Chl a is reduced and that from the red-shifted Fxs is minimum. Therefore, Chl c and the blue-shifted Fxs are the only contributors to the energy transfer pathways under HL and the blue- to red-shifted Fxs energy transfer pathway characteristic of the LL is inactive. We suggest that the light intensity dependent conformational changes of the pigments affects the excitonic coupling of the complexes involved in the emission. The excited state dynamics of Fx probed by time-resolved fs absorption spectroscopy will also be presented.

[1] C. Tselios, Varotsis, C. Evidence for a reversible light-dependent transition in the photosynthetic pigments of diatoms RSC Advances, 23, (2022) 31555-31563.

[2] C. Andreou, Tselios, C., A. Ioannou, A., Varotsis, C. Probing the Fucoxanthin-Chlorophyll a/c-Binding proteins (FCPs) of the Marine diatom Fragilariopsis sp. by Resonance Raman spectroscopy J. Phys. Chem. B , 127 (2023) 9014-9020.

Oxygenic photosynthesis is the light-driven conversion of carbon dioxide to biomass in plants, algae and cyanobacteria. During this process, photosystem II (PSII), a multi-subunit thylakoid membrane protein complex, catalyses the light-driven oxidation of water at Mn₄CaO₅ cluster. The PSII mainly forms a dimer with each monomer containing reaction centre subunits D1 and D2, inner antenna subunits CP43 and CP47, small subunits and extrinsic subunits PsbO, U and V covering and stabilising the Mn₄CaO₅ cluster, which is coordinated by one CP43 residue, two D1 lumenal residues and four residues from D1 C-terminal tail. The structure and function of PSII are well known. However, assembly/disassembly mechanism of the Mn₄CaO₅ cluster is unclear.

We structurally investigated a dimeric PSII intermediate by cryo-EM in native T. elongatus. This PSII intermediate was eluted as the fourth fraction in ion exchange chromatography during PSII purification [1], and here we termed it Peak4 PSII. The Peak4 PSII is a mixture of dimeric PSII complexes containing active PSII with extrinsic subunits at both monomers, inactive PSII lacking extrinsic subunits at both monomers and semi-active PSII lack extrinsic subunits at one monomer. Compared to fully functional PSII, the inactive PSII monomers show missing Mn₄CaO₅ cluster, D1 C-terminal tail and an open channel that reaches the binding site of Mn₄CaO₅ cluster. Thus, we propose that Mn₄CaO₅ cluster assembly results in the folding of D1 C-terminal tail, which then triggers D2 C-terminal tail and PSII lumenal region conformational changes to accept extrinsic subunits. Interestingly, a single oxidation event at D1-His332 imidazole ring were observed. Given that D1-His322 oxidization is an early photodamage event [2], our structures might also reflect early disassembly complexes.

[1] M.M. Nowaczyk, R. Hebeler, E. Schlodder, H.E. Meyer, B. Warscheid, M. Rögner, Psb27, a cyanobacterial lipoprotein, is involved in the repair cycle of photosystem II, Plant Cell 18 (2006) 3121–3131. https://doi.org/10.1105/tpc.106.042671.

[2] R. Kale, A.E. Hebert, L.K. Frankel, L. Sallans, T.M. Bricker, P. Pospíšil, Amino acid oxidation of the D1 and D2 proteins by oxygen radicals during photoinhibition of Photosystem II, Proc. Natl. Acad. Sci. U.S.A. 114 (2017) 2988–2993. https://doi.org/10.1073/pnas.1618922114.

Mitochondrial cytochrome c oxidase (CcO) catalyzes the electron transfer from ferrocytochrome c to O₂ leading to water formation and contributes to the formation of electrochemical proton gradient on inner mitochondrial membrane. The current models of the proton pumping in CcO rely on the existence of a “high-energy” metastable O_H state, which is formed immediately after oxidation of the reduced CcO with oxygen. A part of the energy released in the redox reactions of the CcO catalytic cycle and needed for proton pumping is stored in this O_H state. The O_H is supposed to relax to the resting “as isolated” oxidized state (O) in a time exceeding 200 ms. The catalytic heme a₃-Cu_B center of these two forms should differ in protonation and ligation states and the transition of O_H-to-O is suggested to be associated with a proton transfer into this center. Employing a stopped-flow and UV-Vis absorption spectroscopy, we investigated a proton uptake during the supposed relaxation of O_H. It is shown, using pH indicator phenol red, that from a time when the oxidation of the fully reduced CcO is completed (~25 ms) up to ~10 minutes, there is no uptake of a proton from external medium (pH 7.8). Moreover, the interactions of O and supposed O_H with H₂O₂ (1 mM), resulting in the formation of two ferryl intermediates of the catalytic center, P and F, were very similar both in the kinetics and the amounts of the formed ferryl states. These results together with previous findings suggest that the relaxation of the catalytic center during the O_H-to-O transition is either shorter than 100 ms or there is no difference in the protonation and ligation state of the catalytic sites of O and O_H.

NADH-ubiquinone (UQ) oxidoreductase (complex I) couples electron transfer from NADH to UQ with proton translocation across the membrane. The UQ reduction step is a key part of energy transmission from the site of UQ reaction to the remotely located proton pumping machinery of the enzyme. Although structural studies have identified a long and narrow UQ-accessing tunnel, it remains debatable whether this tunnel model can account for the binding of various ligand molecules (i.e. substrate UQs and inhibitors) to the enzyme. We previously investigated whether a series of oversized UQs (OS-UQs), whose tail moiety is too large to enter and transit the narrow tunnel, can be catalytically reduced by complex I using the native enzyme in bovine heart submitochondrial particles (SMPs) and the isolated enzyme reconstituted into liposomes. Nevertheless, the physiological relevance remains unclear because some amphiphilic OS-UQs were reduced in SMPs but not in proteoliposomes, and investigation of extremely hydrophobic OS-UQs was not feasible in SMPs [1]. In the present study, the accessibility of OS-UQs to the proposed tunnel was evaluated using bovine SMPs fused with liposomes containing extremely hydorophobic OS-UQs. We found that OS-UQs can function as efficient electron acceptors from “native” complex I in SMPs [2]. We also synthesized a phosphatidylcholine-quinazoline hybrid compound (PC-Qz1), in which a quinazoline-type toxophore was attached to the sn-2 acyl chain to prevent it from entering the tunnel and investigated whether this hybrid compound can inhibit complex I. Surprisingly, PC-Qz1 inhibited complex I and suppressed photoaffinity labeling by another quinazoline derivative, [¹²⁵I]AzQ. These studies provide further experimental evidence that challenges the physiological relevance of the prevailing tunnel model.

[1] Uno et al. Oversized ubiquinones as molecular probes for structural dynamics of the ubiquinone reaction site in mitochondrial respiratory complex I. J. Biol. Chem. 295 (2020), 2449-2463.

[2] Ikunishi et al. Respiratory complex I in mitochondrial membrane catalyzes oversized ubiquinones. J. Biol. Chem. 299 (2023), 105001.

Respiratory complex I is a key metabolic enzyme across many respiratory chains, pairing the reduction of NADH and oxidation of a quinone to pump four protons across an energy transducing membrane, maintaining a protonmotive force that drives the cell. The mechanisms of catalysis and regulation of complex I remain contentious, in particular the coupling of proton pumping in the membrane arm to redox reactions in the hydrophilic arm [1][2]. This is in part due to the lack of structures of biochemically characterised intermediates on the complex I catalytic cycle. Paracoccus denitrificans provides a genetically tractable model organism, closely related to the mammalian mitochondrion, from which complex I can be purified and structurally characterised [3]. Mutations have been generated in P. denitrificans complex I which prevent turnover, offering an opportunity to capture a uniform population stalled during a turnover cycle, as opposed to a mixture of intermediates captured under normal turnover conditions. Here we elucidate the structure of a catalytically dead complex I mutant in P. denitrificans, stabilised in nanodiscs, in an attempt to capture a stalled intermediate in the turnover cycle of complex I.

[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] D. Kampjut, L. A. Sazanov, Structure of respiratory complex I – An emerging blueprint for the mechanism, Curr. Opin. Struct. Biol. 74 (2022) 102350.
[3] O.D. Jarman, O. Biner, J.J. Wright, J. Hirst, Paracoccus denitrificans: a genetically tractable model system for studying. respiratory complex I, Sci Rep. 11(1) (2021) 10143.

The most common primary mitochondrial diseases result from mutations of complex I. Inheritance of mutant NDUFS4, thought to support complex I assembly, causes partial complex I deficiency and the severe neuromuscular disease Leigh syndrome. However, even with complete deletion of NDUFS4 residual complex I activity remains in mice. We have investigated the state of assembly of complex I and respiratory supercomplexes isolated from livers of NDUFS4 mutant mice. We confirm holo-complex I only exists in supercomplexes, as do incompletely assembled complex I subassemblies. We determine the structure of supercomplexes containing complex I subassemblies lacking: 1) the N-module (N-less); and 2) entire peripheral arm (membrane domain alone). Thus, we observe stalled complex I assembly intermediates bound to complex III₂ and complex IV. These findings support the view that supercomplexes are early scaffolds of complex I assembly and not a late stage higher-order product forming after all complexes are fully assembled and thus, enhancing supercomplex formation may help mitigate complex I deficiency.

ATP synthase, or Complex V, plays a primary role in the production of ATP in mitochondria; however, it is hypothesized to have other structural and functional roles – specifically in forming and/or regulating mitochondrial permeability transition pore (mPTP) opening. While the dimeric state of Complex V (CV₂) has been proposed to form the mPTP, this would predict the rapid ability of Complex V to reorganize into dimers. However, it remains unknown if this can occur on the acute time scale of mPTP opening. Therefore, the goal was to determine 1) if Complex V dimerization can be acutely induced by calcium overload, 2) if the presence of the CV₂ is correlated with mPTP opening and loss of mitochondrial membrane potential, and 3) if inhibitors of mPTP opening, inhibit dimerization. Mitochondria isolated from rat hearts were added to a respiration media with fuel (glutamate + malate) and incubated with low and high calcium concentrations, while membrane potential and respiration were measured. Samples of mitochondria were aspirated from the respiration chamber, pelleted, solubilized (digitonin), and run on a gradient BN-PAGE gel to allow for the assessment of CV forms. CV₁ and CV₂ were determined by densitometry and percent of CV₁/(CV₁+CV₂) and CV₂/(CV₁+CV₂) were determined. Acute calcium-overload of isolated mitochondria resulted in an increase in the expression of CV₂ and a concomitant decrease in CV­₁: High calcium resulted in an increase in CV₂ (from 35.3±3.2% to 53.1±3.3%) and a decrease in CV₁ (from 64.7±3.2% to 46.9±3.3%), and this calcium overload was associated with loss of mitochondrial calcium and membrane potential. Cyclosporin A, an inhibitor of mPTP opening, prevented loss of mitochondrial calcium and membrane potential, but still resulted in the formation of CV₂, (35.3±3.2% vs 39.9±6.3%). Oligomycin blunted Complex V₂ formation: CV₂ increased only 12.0%, compared to 101.7% in the absence of oligomycin. The adenine nucleotide translocase (ANT) is hypothesized to play a role in mPTP formation, and, intriguingly, inhibition of ANT with carboxyatractoloside (c-state) promoted CV₂ formation by 32%, while Bongkrekic acid (m-state) prevented CV₂ formation, mPTP opening, and loss of membrane potential. These data indicate Complex V can acutely reorganize into dimers in seconds in response to intracellular signals, and that Complex V and ANT interact to form the mPTP.

Activation of brown adipocytes in mammals is reliant on the enhanced activity of respiratory complexes and allows organismal adaptation to cold environments. Comparative biochemical and cryo-EM analyses of respiratory complexes CI:III₂ from thermoneutral (low metabolic activity) and cold-acclimated (high metabolic activity) brown adipose tissue from mice reveal a distinct CI:III₂ assembly in the cold-acclimated mice (termed as type 2) that is absent in the thermoneutral condition (termed as type 1). This cold-inducible type 2 non-canonical CI:CIII₂ supercomplex (SC) structure (type 2) [1] displays a 25° rotation of CIII₂ relative to CI compared to the canonical form type 1, creating a larger void for lipids to occupy the region between them.

Large scale classical MD simulations of a ~2.1 million atom model of CI:III₂ SC in realistic membrane-solvent environment were performed to reveal the molecular basis of the physiological differences between the two states (type 1 and 2). MD simulations of type 2 SC exhibit an increase in CIII stability compared to type 1 owing to the more rotated arrangement of CIII with respect to CI. We find the higher lipid occupancy in the region between the two respiratory complexes to be partly responsible for the enhanced stability of type 2 SCs. Moreover, we show how different lipid species interact with the CI:CIII₂ interface through the simulations of the two different states.

The simulation findings provide a dynamic view into the differences between type 1 and 2 interactions and how the enrichment of type 2 CIII₂ rotated conformation in cold-adapted brown fat tissues can enhance the activity of the respiratory complexes. We suggest a model wherein, under thermoneutral conditions, the energy barrier for transitioning from type 1 to type 2 is higher compared to colder environments.

[1] Y.-Ch. Shin, P. Latorre-Muro, A. Djurabekova, O. Zdorevskyi, Ch. F. Bennett, N. Burger, K. Song, Ch. Xu, V. Sharma, M. Liao, P. Puigserver. Structural basis of respiratory complexes adaptation to cold temperatures. bioRxiv (Pre-Print) (2024)

Succinate dehydrogenase (SDH) is a crucial enzyme that provides a functional link between the Krebs cycle and the respiratory electron transport chain in mitochondria and many microorganisms [1]. The general blueprint for SDH architecture comprises a flavoprotein subunit (SdhA), an iron-sulfur cluster subunit (SdhB), and a transmembrane part, composed of one or more subunits [1].

In Sulfolobus acidocaldarius, a model organism for the Thermoproteota group within the Archaea domain, the SDH was shown to form an unusual assembly, with the membrane anchor replaced by two subunits exhibiting no homology to other known SDHs components [2]. We employed novel computational tools: AlphaFold2 [3] and AlphaFill [4], to predict the structure of SdhABEF and the arrangement of its cofactor chain. According to our results, the SdhABEF is a water-soluble complex with no membrane anchor. Indeed, we have successfully isolated the whole, active complex from the cytoplasmic fraction of the cell lysate. Moreover, we designed the Gateway-harnessing expression vector for S. acidocaldarius to achieve efficient production and purification required for cryo-EM and functional studies.

[1] T.M. Iverson, P.K. Singh, G. Cecchini, An evolving view of complex II-noncanonical complexes, megacomplexes, respiration, signaling, and beyond, J Biol Chem 299 (2023) 104761. https://doi.org/10.1016/j.jbc.2023.104761.

[2] S. Janssen, G. Schäfer, S. Anemüller, R. Moll, A succinate dehydrogenase with novel structure and properties from the hyperthermophilic archaeon Sulfolobus acidocaldarius: Genetic and biophysical characterization, Journal of Bacteriology 179 (1997) 5560–5569. https://doi.org/10.1128/jb.179.17.5560-5569.1997.

[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.

Cytochrome c oxidase catalyzes the 4 electron reduction of O₂ to H₂O, while simultaneously facilitating the translocation of 4 protons across the mitochondrial inner membrane. Recent structural studies have shed new light onto the catalytic cycle of this reaction by capturing the intermediates [1]. Here, we expand on this work by presenting the structures of remaining intermediates with improved resolution.

[1] Kolbe, F. et al. Cryo-EM structures of intermediates suggest an alternative catalytic reaction cycle for cytochrome c oxidase. Nat. Commun. 12, 6903 (2021).

Prevotella bryantii, a strictly anaerobic bacterium, is highly abundant in the rumen microbiome. In P. bryantii, interplay of the Na⁺-translocating NADH:quinone oxidoreductase (NQR) and the quinol:fumarate reductase (QFR) results in NADH:fumarate oxidoreduction in P. bryantii, mediated by the electron carrier menaquinone. Like the NQR from Vibrio cholerae [1], P. bryantii NQR acts as a sodium pump, generating an electrochemical potential during NADH:menaquinone oxidoreduction. In contrast, menaquinol oxidation by P. bryantii QFR does not result in the build-up of a membrane potential [2]. To elucidate the mode of action of NQR and QFR from P. bryantii, we performed homology modelling based on known structures of the complexes [3]. The models revealed putative cofactor-binding sites, interactions with substrates and electron transfer pathways. Interactions of menaquinone with the quinone site of subunit NqrB of P. bryantii were predicted by molecular docking. Comparing the 3D model of P. bryantii with experimentally determined QFR structures from D. gigas and Wolinella succinogenes suggests a divergent mechanism for compensatory, transmembrane proton transport. Our results elucidate the mode of energy generation during NADH:fumarate oxidoreduction by P. bryantii.

[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, 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] L. Schleicher, A. Trautmann, D.P. Stegmann, G. Fritz, J. Gätgens, M. Bott, S. Hein, J. Simon, J. Seifert, J. Steuber, A Sodium-Translocating Module Linking Succinate Production to Formation of Membrane Potential in Prevotella bryantii, Applied and Environmental Microbiology 87 (2021) e01211-21. https://doi.org/10.1128/AEM.01211-21.

[3] J.-L. Hau, L. Schleicher, S. Herdan, J. Simon, J. Seifert, G. Fritz, J. Steuber, Functionality of the Na⁺-translocating NADH:quinone oxidoreductase and quinol:fumarate reductase from Prevotella bryantii inferred from homology modeling, Arch Microbiol 206 (2023) 32. https://doi.org/10.1007/s00203-023-03769-5.

Cellular respiration is essential for aerobic life. A key component of the mitochondrial respiratory chain is complex III (cytochrome bc1 complex), which also associates into supercomplexes. It is one of the proton-translocating respiratory complexes, which converts energy harnessed from the oxidation of food sources into a proton motive force to drive the synthesis of adenosine triphosphate in oxidative phosphorylation. Fully assembled complexes are required to fulfil this function and gene defects that impair the assembly are linked to diseases. The assembly of complex III is still poorly understood. It requires the correct association of catalytic and accessory subunits and the integration of redox cofactors. We will report on the molecular mechanism and structural basis of defined assembly steps of mitochondrial complex III.

Actinobacteria harbour an obligate respiratory supercomplex in which a membrane-bound cytochrome cc subunit connects complexes III and IV to mediate electron transfer throughout the supercomplex. Actinobacteria contain multiple pathogenic species such as Mycobacterium tuberculosis and Mycobacterium leprae. The structural and functional differences, compared to the canonical respiratory chain (RC), makes the supercomplex a promising drug target. Using biochemical, structural and computational approaches we have analysed the known substrate binding sites in the RC supercomplex from M. smegmatis, which has high homology to the M tuberculosis RC supercomplex, in order to obtain better understanding of their function. We also identify an inhibitor with possible drug potential aimed at this RC protein complex.

In mammals, the mitochondrial respiratory chain (MRC) complexes I, III and IV can associate in supercomplexes (SCs) and respirasomes, whose biogenetic regulation and functional properties are defined by the tissue-specific expression of three complex IV COX7A subunit isoforms, namely COX7A1, COX7A2 and COX7A2L (SCAFI). Previous studies from our lab showed the coexistence of two separated MRC organizations in human cells and post-mitotic tissues: the bioenergetically more efficient C-MRC, defined by COX7A1/2, and the S-MRC, defined by SCAFI. In human cultured HEK293T cells and primary fibroblasts, the prevalence of each MRC organization was reversibly regulated by the activation state of the pyruvate dehydrogenase complex (PDC), a major regulator of the metabolic switch between glycolysis and oxidative phosphorylation (OXPHOS). Activated PDC promoted the COX7A1/2-dependent C-MRC organization and OXPHOS, whereas inactivated PDC stimulated the SCAFI-dependent S-MRC upon metabolic rewiring towards glycolysis. This data suggested a possible pathophysiological involvement of SCAFI and the S-MRC in metabolic disorders and cancer. To determine the impact of the structural loss of PDC on the S-MRC organization, we used the MIA PaCa-2 pancreatic tumor cell line depleted of the PDHA1 subunit (MP2-PDHA1-KO cells). Unexpectedly, the absence of PDHA1 led to increased C-MRC with reduced levels of SCAFI and the S-MRC, which were recovered after overexpression of wild-type PDHA1. Similarly, overexpression of a non-phosphorylatable PDHA1 version, resulting in stable expression of hyperactive PDC (MP2-PDHA1^AAA cells) neither restored SCAFI levels, but instead promoted the C-MRC organization. Our results suggest that the upregulation of the C-MRC can be modulated by metabolic pathways not necessarily converging on the PDC. In contrast, the up-regulation of the S-MRC organization would exclusively depend on both the presence and phosphorylation-mediated inactivation of the PDC.

Ref: Fernández-Vizarra E.; López-Calcerrada S.; et al; Ugalde C. 2022. Two independent respiratory chains adapt OXPHOS performance to glycolytic switch. Cell Metabolism 34-11, 1792-1808. Doi: 10.1016/j.cmet.2022.09.005

Several human diseases, from cancer to neurodegeneration, are associated with excessive mitochondrial fission. The mitochondrial division inhibitor (Mdivi-1) has been tested as a therapeutic target for inhibiting fission-related protein Dynamin-like protein 1 (Drp1) [1] [2]. A study in 2017 raised a debate about significant off-target effects such as inhibiting complex I [3].

Here, we show that the effects of Mdivi-1 on mitochondrial morphology and function, ultimately leading to cellular dysfunction of neurons, are mechanistically based on direct complex I inhibition at the I_Q site. This leads to destabilization of the complex I and supercomplexes, increased ROS production and ultimately reduced mitochondrial ATP. Furthermore, the calcium homeostasis of cells is dampened, which in the long run attenuates the activity of neurons. Given the results presented here, a putative therapeutic application of Mdivi-1 will need to be reconsidered in terms of dose- and time-dependent effects on mitochondrial energy metabolism, particularly OXPHOS activity.

[1] Baek SH, Park SJ, Jeong J in, et al. Inhibition of Drp1 Ameliorates Synaptic Depression, Aβ Deposition, and Cognitive Impairment in an Alzheimer's Disease Model. J Neurosci. 2017;37(20):5099-5110.

[2] Rappold PM, Cui M, Grima JC, et al. Drp1 inhibition attenuates neurotoxicity and dopamine release deficits in vivo. Nat Commun. 2014;5:5244.

[3] Bordt EA, Clerc P, Roelofs BA, et al. The Putative Drp1 Inhibitor mdivi-1 Is a Reversible Mitochondrial Complex I Inhibitor that Modulates Reactive Oxygen Species. Dev Cell. 2017;40(6):583-594.

Cytochrome bd oxidases are membrane proteins found only in prokaryotes which catalyze the reduction of O₂ by two equivalents of quinol and contribute to the proton motive force required for ATP synthesis by taking the four protons required for O₂ reduction from the cytoplasmic side and releasing protons from quinol oxidation to the periplasmic side of the membrane^[1]. They play a crucial role in the tolerance of bacteria to oxidative and nitrosative stress conditions^[2] and the resistance to antibiotics^[3], although the molecular mechanisms are not fully understood.

The oxygen reductase activity and inhibition of cytochrome bd oxidases can be characterized by means of Protein Film Voltammetry. We have immobilized the enzymes on 3D gold nanoparticle electrode modified with self-assembled monolayers of thiols and lipids as described previously^[4]. The gold nanoparticles facilitate the electron transfer from the electrode to the cofactors located deep inside the enzyme. On this basis, the electrochemical signature of the cytochrome bd oxidase from the anaerobic sulfate-reducing bacterium Solidesulfovibrio fructosivorans in the presence of various quinones as electron donor and small uncharged signalling molecules such as NO was successfully obtained. The redox potentials of the heme cofactors of the protein were determined by potentiometric titrations in an electrochemical thin layer cell. The results are compared to data previously reported on the cytochrome bd oxidases from Escherichia coli, Geobacillus thermodenitrificans and Corynebacterium glutamicum^[5].

[1] V.B. Borisov, S.A. Siletsky, A. Paiardini, D. Hoogewijs, E. Forte, A. Giuffrè, R.K. Poole, Antiox. Redox Signal., 34 (2021) 1280-1318.

[2] A. Giuffrè, V.B. Borisov, M. Arese, P. Sarti, E. Forte, Biochim. Biophys. Acta, Bioenerg., 1837 (2014) 1178-1187.

[3] P. Lu, A.H. Asseri, M. Kremer, J. Maaskant, R. Ummels, H. Lill, D. Bald, Sci. Rep., 8 (2018) 2625.

[4] I. Makarchuk, A. Nikolaev, A. Thesseling, L. Dejon, D. Lamberty, L. Stief, A. Speicher, T. Friedrich, P. Hellwig, H.R. Nasiri, F. Melin, Electrochim. Acta, 381 (2021) 138293.

[5] A. Nikolaev, S. Safarian, A. Thesseling, D. Wohlwend, T. Friedrich, H. Michel, T. Kusumoto, J. Sakamoto, F. Melin, P. Hellwig, Biochim. Biophys. Acta, Bioenerg. 1862 (2021), 148436.

Mitochondrial respiratory complexes interact and assemble into supramolecular structures called supercomplexes. The most conserved interspecies interaction is that between complex I (CI) and complex III (CIII), resulting in biochemically and structurally well-known supercomplexes such as I+III₂ or I+III₂+IV (N-respirasome). Several functions have been proposed for this interaction, among them: substrate channeling, decrease in reactive oxygen species production or scaffolding for CI assembly. The biochemical and physiological consequences of the absence of the CI-CIII superassembly are still an unresolved question in the field. To investigate in depth the role of the interaction between complexes I and III in bioenergetics and metabolism, we destabilized most of CI-CIII superassembly in cultured mouse fibroblasts by two different and complementary approaches. To do this, we performed amino acid substitutions in two subunits of CIII involved in the interaction with CI, UQCRQ and UQCRC1. With this approach we aim to shed light on the role of the very conserved CI-CIII superassembly in mitochondrial biology, a question that has been the subject of intense debate for more than two decades.

Respiratory complex I serves as a main entry point of electrons derived from oxidative catabolism into respiratory chains and therefore plays a crucial role in the cellular energy metabolism. In canonical ubiquinone-dependent respiration, it couples the exergonic oxidation of NADH to the endergonic translocation of protons against a proton gradient across the cytosolic membrane, which ultimately drives ATP synthesis. It is the current consensus that the reduction of ubiquinone by NADH drives the translocation of four protons per oxidized NADH [1]. Anaerobic microorganisms, however, predominantly contain menaquinone with a considerably lower midpoint redox potential than ubiquinone (‑75 mV vs. ≈100 mV) [2]. Therefore, the resulting Gibbs free energy difference does not allow for the translocation of four protons. Moreover, many anaerobic organisms also produce reduced ferredoxin and NADPH instead of NADH through alternative tricarboxylic acid cycle enzymes [3]. This raises the question whether NADPH and reduced ferredoxin can serve as electron donors for menaquinone-dependent respiratory complexes I and how many protons are translocated in the process.

To study respiratory complex I in these organisms, we enriched the electron-transferring, peripheral domain of the enzyme from the strictly anaerobic model organism Geobacter metallireducens and observed a clear preference for NADPH of the purified enzyme complex. Homologies to electron-bifurcating FeFe-hydrogenases and the identification of additional iron-sulfur clusters not present in canonical respiratory complex I, suggests that menaquinone reduction and proton translocation is facilitated by flavin-based electron confurcation.

[1] A.A. Stuchebrukhov, T. Hayashi, Single protonation of the reduced quinone in respiratory complex I drives four‐proton pumping, FEBS Letters. 597 (2022) 237–245. doi:10.1002/1873-3468.14518.

[2] W. Nitschke, D.M. Kramer, A. Riedel, U. Liebl, From naphtho- to benzoquinones - (R)evolutionary reorganisations of electron transfer chains, Photosynthesis: From Light to Biosphere. (1995) 945–950. doi:10.1007/978-94-009-0173-5_225.

[3] A.S. Galushko, B. Schink, Oxidation of acetate through reactions of the citric acid cycle by Geobacter sulfurreducens in pure culture and in Syntrophic Coculture, Archives of Microbiology. 174 (2000) 314–321. doi:10.1007/s002030000208.

In cell respiration, tyrosyl radicals play a crucial role in the formation of ferry intermediates which are stabilized for efficient energy conservation and proton translocation. The properties of the P and F ferryl intermediates in the reactions of oxidized cytochrome c oxidase (CcO) from P. denitrificans with H₂O₂ were investigated by monitoring the 244-nm resonance Raman spectra of tyrosine-280, tyrosine-167 and tryptophan-267 mutants. At early times in the reaction, the RR spectra of the reactions of Tyrosine-280 and wild type are nearly identical and showed the formation of bands characteristic of tyrosine/tyrosinate and tryptophan residues. The decay of these transient species to the oxidized form of the enzyme is completed within 15 minutes after mixing with H₂O₂. The reaction of MV CcO Y280 with O₂ was also investigated by Uv-Raman spectroscopy. The time evolution of the transient species formed will be presented.

[1] E. Pinakoulaki, V. Daskalakis, T. Ohta, O-H Richter, K. Budiman, T. Kitagawa, B. Ludwig, C. Varotsis The protein effect in the structure of two ferryl-oxo intermediates at the same oxidation level in the heme copper binuclear center of cytochrome c oxidase J. Biol. Chem., 288 (2013) 20261-20266.

[2] V. Daskalakis, S.C. Farantos, C. Varotsis, Assigning vibrational spectra of ferryl–oxo intermediates of cytochrome c oxidase by periodic orbits and molecular dynamics, J. Am. Chem. Soc., 130 (2008) 12385–12393.

[3] E. Pinakoulaki, V. Daskalakis, C. Varotsis The origin of the FeIV=O intermediates in cytochrome aa₃ oxidase Biochimica et Biophysica Acta (BBA)-Bioenergetics, 1817 (2012) 552-557.

In methanogenic archaea the energy-converting N5-methyltetrahydromethanopterin:
coenzyme M methyltransferase catalyzes an exergonic methyl-transfer reaction to pump sodium-ions across the cell-membrane, forming a sodium-ion gradient that drives ATP-synthesis. The reaction proceeds in two steps, starting with a methyl-transfer from methyl-tetrahydromethanopterin to 5-hydroxybenzimidazole-cob(I)amid (Factor III), forming the methyl-cob(III)amid. In the second step the methyl-group is transferred to CoM to form methyl-CoM, thereby pumping sodium-ions. Methyl-CoM is further reduced to methane and CoM, completing the methanogenesis pathway. Interestingly, the endergonic reverse reaction can be driven by a sodium-gradient. The reversibility is physiologically important for methylotrophic methanogenic archaea, as they need to oxidize one methyl-group per three methane molecules formed.

In our study we elucidated the cryo-EM structure of the full Mtr complex of a methylotrophic methanogen. The obtained map allowed us to model all subunits, including the methyltransferase MtrH and the cobamid-binding domain of MtrA. Gentle isolation procedure from the native organism revealed a previously unidentified zinc-binding site that could change the way of how we imagine the functionality of the complex. Our results enhance our understanding how this ancient and unique way of energy conservation works.

Respiratory complex I is a 1 MDa membrane protein complex that couples electron transfer from NADH to ubiquinone to the pumping of protons across the mitochondrial inner membrane and plays a central role in aerobic energy metabolism. Although high resolution structures of complex I from several organisms [1] and details of its assembly process [2] have been revealed by cryo-EM studies, the structural basis of the coupling mechanism is not clear [3,4].

A recent high-resolution structure of complex I from the thermophilic filamentous fungus Chaetomium thermophilum [5] indicated the coexistence of two conformations (state 1 and state 2), characterized by a small twisting motion of the peripheral arm relative to the membrane arm. The two states show rearrangements of structural elements in the electron transfer pathways, similar to those found in the “open” and “closed” states of mammalian complex I, respectively. We have now determined structures of C. thermophilum complex I in presence of the substrates NADH and ubiquinone-1. Both substrates are visible in the cryo-EM maps, and preliminary analyses indicate that state 1 and state 2 are reproduced in this dataset, with an increased proportion of state 2.

[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] E. Laube, J. Schiller, V. Zickermann, J. Vonck, Using cryo-EM to understand the assembly pathway of respiratory complex I, Acta Crystallogr. D Struct. Biol., 80 (2024) 159-173.

[3] V. Kravchuk, O. Petrova, D. Kampjut, A. Wojciechowska-Bason, Z. Breese, L.A. Sazanov, A universal coupling mechanism of respiratory complex I, Nature, 609 (2022) 808-814.

[4] D.N. Grba, J.J. Wright, Z. Yin, W. Fisher, J. Hirst, Structural basis of a regulatory switch in mammalian complex I, bioRxiv, (2023) https://doi.org/10.1101/2023.12.14.571638.

[5] E. Laube, J. Meier-Credo, J.D. Langer, W. Kühlbrandt, Conformational changes in mitochondrial complex I of the thermophilic eukaryote Chaetomium thermophilum, Science Advances, 8 (2022) adc9952.

Respiratory complex I is a key metabolic enzyme in mammalian mitochondria that harnesses the energy from NADH oxidation and ubiquinone reduction to generate the proton motive force for ATP synthesis. In ischemic conditions, complex I switches from an active state that is capable of rapid, reversible catalysis into a dormant (deactive) state that protects upon reoxygenation, preventing ROS production by reverse electron transfer (RET, Δp-driven ubiquinol:NAD⁺ oxidoreduction) and minimizing oxidative damage [1]. The molecular mechanism of this protective switch is unknown. Here, we investigate the mechanism of the deactive transition using a combined structural, biophysical, and biochemical approach in complex I-containing proteoliposomes (CI-PLs). These modular CI-PLs are fully characterizable, display high rates of complex I catalysis and using different combinations of partner enzymes, can catalyze NADH-driven ATP synthesis or NAD⁺ reduction by RET, both mediated by a substantial proton-motive force [2,3]. Complex I in proteoliposomes can be switched between different regulatory states, providing a platform to investigate the conformational changes associated with the deactive transition. We determine the structure of complex I, isolated from ischemic heart tissue (Bos taurus) and embedded in proteoliposomes, at high-resolution, allowing identification of the structural states that are present during the deactive transition of mammalian complex I. Precise biochemical characterization allows assignment of these structural states and to define the mechanisms of the transitions between them. By implementing a versatile membrane system to unite structure and function, we define the molecular basis of the deactive transition of mammalian complex I, providing important insights into complex I catalysis and regulation.

[1] E.T. Chouchani, V.R. Pell, A.M. James, L.M. Work, K. Saeb-Parsy, C. Frezza, T. Krieg, M.P. Murphy, A unifying mechanism for mitochondrial superoxide production during ischemia-reperfusion injury, Cell Metab 23 (2016) 254–263.

[2] O. Biner, J.G. Fedor, Z. Yin, J. Hirst, Bottom-Up Construction of a Minimal System for Cellular Respiration and Energy Regeneration, ACS Synth Biol 9 (2020) 1450–1459.

[3] J.J. Wright, O. Biner, I. Chung, N. Burger, H.R. Bridges, J. Hirst, Reverse Electron Transfer by Respiratory Complex I Catalyzed in a Modular Proteoliposome System, J Am Chem Soc 144 (2022) 6791–6801.

Cellular energy metabolism includes all the metabolic pathways involved in the production of ATP and in NADH turnover. The ATP/ADP and NADH/NAD+ ratios are therefore important parameters regulating cellular metabolic activity. In aerobic cells, mitochondria are the main producers of cellular ATP through the Krebs cycle and the oxidative phosphorylation (OXPHOS). Quinones are lipophilic molecules embedded in the inner mitochondrial membrane (Q₆ in the yeast Saccharomyces cerevisiae) that transfer the electrons within OXPHOS from the dehydrogenases to complex III. Their redox state depends on the dehydrogenases substrates availability and on the presence of oxygen. We thus wondered how does the quinones redox state respond when the OXPHOS activity is modulated and if this parameter could be a potential marker to characterize mitochondrial function.

Based on previous works on plants [1,2], we developed an indirect electrochemical method to monitor in real-time the redox state of quinones using a short-length chain species (Q₂) as a redox mediator. This measurement is associated with the one of the mitochondrial oxygen consumption detected by a Pt Clark electrode.

Our results on isolated mitochondria from S. cerevisiae have revealed different respiratory chain activities and reduction levels of quinones with several respiratory substrates. In the phosphorylating state, while respiration is stimulated for all the substrates, the redox state of quinones varies depending on the respiratory substrate. Our results clearly show an implication of the Krebs cycle function [3] in the modulation of the response of quinones redox state. The analysis of the mitochondrial quinones redox state thus allows the analysis of the mitochondrial metabolic activity.

[1] A.L. Moore, I.B. Dry, J.T. Wiskich, Measurement of the redox state of the ubiquinone pool in plant mitochondria, FEBS Letters 235 (1988) 76–80.

[2] G. Longatte, A. Sayegh, J. Delacotte, F. Rappaport, F.-A. Wollman, M. Guille-Collignon, F. Lemaître, Investigation of photocurrents resulting from a living unicellular algae suspension with quinones over time, Chem. Sci. 9 (2018) 8271–8281.

[3] M. Rigoulet, J. Velours, B. Guerin, Substrate-level phosphorylation in isolated yeast mitochondria, European Journal of Biochemistry 153 (1985) 601–607.

Ubiquinone (UQ) is an essential player in the respiratory electron transfer system. In Saccharomyces cerevisiae strains lacking the ability to synthesize UQ₆, exogenously supplied UQs can be taken up and delivered to mitochondria through an unknown mechanism, restoring the growth of UQ₆-deficient yeast in non-fermentable medium. Since elucidating the mechanism responsible may markedly contribute to therapeutic strategies for patients with UQ deficiency, many attempts have been made to identify the mechanism involved in UQ trafficking in the yeast model. However, definite experimental evidence of the direct interaction of UQ with a specific protein(s) has not yet been identified. To get insight into intracellular UQ trafficking via a chemistry-based strategy, we synthesized a hydrophobic UQ probe (pUQ5), which has a photoreactive diazirine group attached to a five-unit isoprenyl chain and a terminal alkyne to visualize and/or capture the labeled proteins via click chemistry. pUQ5 successfully restored the growth of UQ₆-deficient yeast (Δcoq2) on a non-fermentable carbon source, indicating that this synthetic UQ was taken up and delivered to mitochondria and worked as a UQ substrate of respiratory enzymes. Through photoaffinity labeling of the mitochondria isolated from Δcoq2 yeast cells cultured in the presence of pUQ5, we identified many labeled proteins, including voltage-dependent anion channel 1 (VDAC1) and cytochrome c oxidase subunit 3 (Cox3). Notably, yeast strains lacking COQ2 and VDAC1 genes exhibited a significant reduction in growth on the non-fermentable medium supplemented with exogenous UQ₆, suggesting the involvement of VDAC1 in facilitating the uptake of exogenous UQ₆ into mitochondria. Moreover, careful proteolytic analyses of the labeled Cox3 revealed that pUQ5 binds to the region Gly86–Arg164, which covers TMH3 adjacent to Cox1 and contributes to the interaction with the bound phospholipids. Our study presents the first trial, via chemical biology techniques, for identifying the key players responsible for uptake of exogenous UQ and its accumulation in mitochondria.

The selective reduction of oxygen to water is crucial to life and a central process in aerobic organisms. It is catalyzed by several different enzymes, including cytochrome c oxidases, found for example in mammals, and cytochrome bd oxidases that are solely present in prokaryotes. These essential enzymes also play a crucial role in protection against oxidative stress, in virulence, adaptability and antibiotics resistance. Electrochemical and spectroscopic studies allow obtaining information on the oxygen reduction, coupled protonation processes, and the interaction with small molecules that rule the signaling processes in the biological cell, including NO. [1,2]

Here we present the electrocatalytic study of these membrane proteins immobilized on nanostructured surfaces. Different modes of immobilization have been probed by infrared spectroscopy and the effect of the presence of lipids on the electrochemical reactivity of the membrane proteins analyzed. Side directed mutants at position D58 and nearby residues reveal that this residue is a crucial part of the proton path to the active site. It can be shown that the NO release kinetcs is associated with proton uptake and thus with the oxygen reaction.

[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’ Biochim. Biophys. Acta (2021) 1862, 148436.

[2] Makarchuk, I.; Kägi, J.; Gerasimova, T.; Wohlwend, D.; Melin, F.; Friedrich, T.; Hellwig, P. ‘pH-dependent kinetics of NO release from E. coli bd-I and bd-II oxidase reveals involvement of Asp/Glu58B. Biochim Biophys Acta Bioenerg. (2023) 1864(2):148952.

Mitochondria play a pivotal role in regulating cellular metabolism, and thus, they are involved in determining cell fate. The inner mitochondrial membrane (IMM) is an extremely tight barrier, and consequently, all signaling molecules generated within the mitochondria must be transported through the IMM. The mitochondrial permeability transition pore (mPTP) is a transient increase in the permeability of the IMM to ions. Over the past decade, there has been a growing interest in the mitochondrial F₁F_O ATP synthase with regard to the mPTP. Moreover, ATP dimerization plays a central role in the formation of the IMM in cristae. The c-channel model posits that the pore is a state of an open channel through the c-ring when the F₁-head is dissociated from the F_O-subcomplex. The opening of the mPTP results in ion influx into the matrix and swelling of the mitochondria due to subsequent water influx [1]. This indicates that the induction of mPTP opening results in the dissociation of F₁ and F_O ATP synthase subcomplexes, which consequently affects the cristae architecture. In this project we used advanced techniques including super-resolution microscopy for cristae imaging, tracking and localization microscopy (TALM), and Blue Native PAGE to investigate the spatiotemporal and macromolecular localization of ATP synthase and subcomplexes during mPTP opening.

[1] P. Bernadi, C. Gerle, A. P. Halestrap, E. A. Jonas, J. Karch, N. Mnatsakanyan, E. Pavlov, S. S. Sheu and A. A. Soukas, Identity, structure, and function of the mitochondrial permeability transition pore: controversies, consensus, recent advances, and future directions, Cell Death Differ, 30 (2023) 1869–1885.

Human ATP synthase is made of 29 subunits of 18 types, including the inhibitor protein IF₁. ATP6 and ATP8 are encoded in mitochondrial DNA, with the rest encoded in the nucleus. Assembly of the enzyme involves intermediate modules representing (i) the F₁-catalytic domain (α₃β₃γδε plus inhibitor protein IF₁), (ii) the peripheral stalk (PS; subunits OSCP, F₆, b and d), plus the membrane "wedge" subunits e, f and g [1,2], and (iii) the membrane bound c₈-rotor ring [3]. They form the key intermediate F₁-IF₁-c₈-PS [4]. Then subunits ATP6 and ATP8 are inserted between the c₈-ring and the wedge with subunit j bound to ATP6, forming a proton pathway coupling the proton motive force to ATP synthesis. Finally, monomeric complexes dimerize, subunit k is added, leading to rows of dimeric enzymes. Here, we show that the F₁-domain plus IF₁ assembles in the absence of both the PS and c₈-ring. Without either α- or β-subunits, the central stalk (CS; subunits γ, δ and ε) assembles from the γ-subunit and a preformed δε-complex. With c-subunit present, a γδε-c₈ rotor complex forms. The F₁-domain is completed with the assistance of three assembly factors ATPAF1, ATPAF2 and FMC1, either by the insertion of the CS into a preformed α₃β₃-ring or by building the α₃β₃-ring around the γ-subunit in the preformed CS.

[1] 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. Proc. Natl. Acad. Sci. USA 115 (2018), 2988-2993.

[2] J. He, J. Carroll, S. Ding, I.M. Fearnley, M.G. Montgomery, J.E. Walker, Assembly of the peripheral stalk of ATP synthase in human mitochondria. Proc. Natl. Acad. Sci. USA 117 (2020), 29602-29608.

[3] J. Carroll, J. He, S. Ding, I.M. Fearnley, J.E. Walker, TMEM70 and TMEM242 help to assemble the rotor ring of human ATP synthase and interact with assembly factors for complex I. Proc. Natl. Acad. Sci. USA 118 (2021),e2100558118.

[4] J. He, H.C. Ford, J. Carroll, S. Ding, I.M. Fearnley, J.E. Walker, Persistence of the mitochondrial permeability transition in the absence of subunit c of human ATP synthase. Proc. Natl. Acad. Sci. USA 114 (2017), 3409-3414.

The energy stored in the proton-motive force across the specific membrane of almost all eubacteria, thylakoids, or mitochondria is used by the F₁F_O-ATP-synthase to synthesize ATP (Mitchell, 1967; Boyer, 1997). The F₁F_O-ATP-synthase consists of two connected sectors, F₁ and F_O. Identification by MS/MS of the F₁F_O-ATP-synthase subunits in Ustilago maydis showed the whole set of subunits reported (Esparza-Perusquía, 2017); from these, e and g are the dimerizing subunits. The ATPase activity of the dimer was 9-times higher than the monomer indicating that interface monomer-monomer plays a role in the activity of the F₁F_O-ATP-synthase dimer. In the mitochondria, the ATPase activity of the F₁F_O-ATP-synthase is prevented by the regulatory subunit IF1. Previous studies show that IF1 doesn’t play an important role in the dimerization of F₁F_O-ATP-synthase (Nakamura, 2013). Although IF1 (Inh1) does not play a role in the dimerization of F₁F_O-ATP-synthase, no studies on ATPase dimer activity have been performed. In U. maydis the elimination of the gene codifying the Inh1 subunit didn’t affect the cell growth, glucose consumption, and biomass production in the mutant strain. Ultrastructure and fluorescence analysis show that the size, the shape of the ridges, the lattice, and the distribution of the mitochondria were like that of the wild-type strain. The ΔY_m, ATP synthesis, and oxygen consumption in wild-type and Inh1Δ strains had similar values. Kinetic analysis of ATPase activity of complex V in permeabilized mitochondria showed similar values of V_max and K_m for both strains, and no effect of pH was observed. Interestingly, the dimeric state of complex V occurs in the mutant strain, indicating that this subunit is not essential for dimerization. ATPase activity of the isolated monomeric and dimeric forms of complex V indicated V_max values 4-times higher for the Inh1Δ strain than for the WT strain, suggesting that the absence of Inh1 subunit increased ATPase activity, and supporting a regulatory role for this protein; however, no effect of pH was observed (Romero-Aguilar, 2021). The deletion of gen codifying subunit g in Saccharomyces cerevisiae induces a decrease in dimer amount and modifies the mitochondrial architecture (Paumard, 2002). In U. maydis elimination of g subunit does not prevent dimerization of complex V in the inner membrane. However, the ATPase activity of the dimer is low in contrast to the monomer, suggesting that the g subunit is not essential for dimerization, but it could have an important role in the activity of the enzyme (Esparza-Perusquía, 2023).

Differently from yeast and mammals, Drosophila melanogaster does not show a mitochondrial permeability transition (PT), a process caused by the opening of the so-called permeability transition pore (PTP). The PTP is defined as a Ca²⁺-activated, high-conductance and unselective channel with a maximal conductance of 1.2 nS that allows ions and solutes up to 1.5 kDa to equilibrate across the inner membrane. PTP openings play a role in both Ca²⁺ homeostasis and cell death initiation. Conversely, Drosophila PTP appears to be specialized and operate uniquely as a selective Ca²⁺-release channel (CrC), that does not induce mitochondrial swelling and cell death. ATP synthase was recently demonstrated to mediate the PT in mammals and yeast and to generate a peculiar 53 pS-channel in Drosophila that could represent the CrC. Genetic studies showed that the ablation of ATP synthase “accessory subunits” e and g dramatically affects PT occurrence in mammals and yeast, suggesting a primary role of these small proteins in PTP formation. To shed light on the roles of the two subunits in Drosophila, we generated knock-down (KD) lines for genes encoding either subunit e (ATPsynE) or g (ATPsynG) of ATP synthase. In vivo ubiquitous downregulation of each subunit causes a dramatic arrest in fly development at larval stage, impairs the dimerization and oligomerization states of ATP synthase and decreases mitochondrial respiration, yet the total amount of ATP is unaltered. Strikingly, the sensitivity to Ca²⁺ is decreased in both ATPsynE and ATPsynG KD mitochondria, which require higher matrix Ca²⁺ loads (1.5-fold and 3-fold, respectively) to induce the CrC. Altogether, our results confirm a key role of these two proteins in the formation of Drosophila channel and suggest that the phenotype of KD flies is not entirely due to bioenergetic defects, but may also partially arise from a CrC-related Ca²⁺ dysregulation.

The mitochondrial protein IF1 is the natural inhibitor of the ATP synthase and binds the catalytic domain during the enzyme hydrolytic activity. In many tumors, IF1 is upregulated and acts as a pro-oncogenic factor through different mechanisms [1, 2]. Its overexpression was recently shown to promote the IF1 interaction in cancer cells with the ATP synthase OSCP (oligomycin sensitivity conferring protein) subunit during oxidative phosphorylation [3].

We identified two IF1 mutant forms, K57N and K57Q of the human mature protein, that are recurrent in aggressive and therapy-resistant lung cancers (i.e. non-small cell lung cancer, NSCLC). The transient and stable re-insertion of the ATP5IF1 gene in IF1 KO HeLa cells allowed us to overexpress the wild-type or mutant IF1 proteins and was instrumental for dissecting their effects.

IF1 mutants displayed an increased colony formation compared to wild-type IF1-expressing HeLa cells in soft agar. These IF1 mutants did not affect proliferation, but altered the sensitivity of the PTP opening, causing resistance to cell death. These changes are mediated by the interaction of IF1 with the OSCP subunit, as shown by immunoprecipitation in wild-type and mutant IF1-expressing cells.

Overall, our results suggest that the IF1 mutants found in lung patient biopsies might favor tumor growth by protecting cancer cells from PTP-dependent apoptosis.

[1] G. Solaini, G. Sgarbi, A. Baracca, The F1Fo-ATPase inhibitor, IF1, is a critical regulator of energy metabolism in cancer cells, Biochem Soc Trans 49 (2021) 815–827.

[2] C. Gatto, M. Grandi, G. Solaini, A. Baracca, V. Giorgio, The F1Fo-ATPase inhibitor protein IF1 in pathophysiology, Front Physiol 13 (2022) 917203-14.

[3] C. Galber, S. Fabbian, C. Gatto, M. Grandi, S. Carissimi, M. J. Acosta, G. Sgarbi, N. Tiso, F. Argenton, G. Solaini, A. Baracca, M. Bellanda, V. Giorgio, The mitochondrial inhibitor IF1 binds to the ATP synthase OSCP subunit and protects cancer cells from apoptosis, Cell Death Dis 14 (2023) 54-73.

Mitochondrial F_OF₁ ATP synthase couples ATP synthesis or hydrolysis to transmembrane proton transport. The primary function of the enzyme is ATP synthesis driven by protonmotive force (pmf) generated by the respiratory chain. If pmf is low, F_OF₁ works as a proton-pumping ATPase. In yeast, ATPase activity of F_OF₁ can be suppressed by inhibitory proteins Inh1p and Stf1p, and MgADP (non-competitive ADP-inhibition). These mechanisms may help the cell to preserve ATP upon mitochondrial membrane de-energization. Nonetheless, no direct evidence was presented to support this hypothesis.

We found that a point mutation Q263L in β subunit of Saccharomyces cerevisiae F_OF₁ attenuates ADP-inhibition without major effect on the rate of ATP production by mitochondria. We used this mutation to test the role of ADP-inhibition in the living yeast cell. The mutant strain grew slower and had a longer lag period preceding exponential growth phase after starvation. However, in yeast lacking mitochondrial DNA (ρ⁰) the effect was reversed: the βQ263L ρ⁰ mutant grew faster than the wild-type ρ⁰ yeast [1].

We also investigated the physiological role of Inh1p and Stf1p, two yeast homologs of mammalian ATP synthase inhibitory factor IF₁. We examined the cells in which Inh1p or Stp1p were fused to green fluorescent protein (GFP) and found that cells increase the concentration of both Inh1-GFP and Stf1-GFP in the post-diauxic phase. During this phase, the cells formed two subpopulations distinct in Inh1p-GFP and Stf1p-GFP concentration. Upon exit from the post-diauxic phase the cells with high level of Inh1-GFP started growing earlier than cells devoid of Inh1-GFP. However, the genetic deletion of these two factors did not increase the lag period necessary for stationary phase yeast cells to start growing after reinoculation into the fresh medium [2].

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

[1] A. Lapashina, N. Kashko, V. Zubareva, K. Galkina, O. Markova, D. Knorre, B. Feniouk, Attenuated ADP-inhibition of F_OF₁ ATPase mitigates manifestations of mitochondrial dysfunction in yeast, BBA Bioenergetics, 1863 (2022) 148544.

[2] K. Galkina, V. Zubareva, N. Kashko, A. Lapashina, O. Markova, B. Feniouk, D. Knorre, Heterogeneity of starved yeast cells in IF1 levels suggests the role of this protein in vivo, Frontiers in Microbiology, 13 (2022) 816622.

The F₁F_O-ATP synthase synthesizes more than 90 % of cellular ATP, but it can also hydrolyze it under particular conditions. This hydrolytic activity can be adverse for organisms as depletion of ATP will cause cellular death. Because of this, three regulatory subunits have emerged: IF₁ in mitochondria, ε in most bacteria, and ζ in α-proteobacteria. The ε subunit functions as an ATP sensor, in which when in the cytoplasm of bacteria if the concentration of ATP is low, ε will have a conformational change, where its C-terminus will adopt an extended conformation and inhibit the ATPase activity of the enzyme. The mitochondrial IF₁ functions as a pH sensor; when mitochondrial matrix pH is basic, it is in a non-inhibitory tetramerized conformer. When the pH is slightly acid, it adopts an inhibitory dimeric conformer. The z subunit is known to bind ATP, but how the binding of ATP in the regulatory mechanism is still unknown[1]. More so, as we were determining the structure by NMR of ζ from the α-proteobacteria Sinorhizobium meliloti (PDB ID 7VKV)[2], we found in our HSQC spectra that at different pH, there were chemical shifts. Here, we explore by ¹H, ¹⁵N-HSQC the pH sensibility of the ζ subunit from Sinorhizobium meliloti.

[1] P. Serrano, M. Geralt, B. Mohanty, K. Wüthrich, NMR Structures of α-Proteobacterial ATPase-Regulating ζ-Subunits, J Mol Biol 426 (2014) 2547–2553. https://doi.org/doi:10.1016/j.jmb.2014.05.004.

[2] F. Mendoza-Hoffmann, L. Yang, D. Buratto, J. Brito-Sánchez, G. Garduño-Javier, E. Salinas-López, C. Uribe-Álvarez, R. Ortega, O. Sotelo-Serrano, M.Á. Cevallos, L. Ramírez-Silva, S. Uribe-Carvajal, G. Pérez-Hernández, H. Celis-Sandoval, J.J. García-Trejo, Inhibitory to non-inhibitory evolution of the ζ subunit of the F1FO-ATPase of Paracoccus denitrificans and α-proteobacteria as related to mitochondrial endosymbiosis, Front. Mol. Biosci. 10 (2023). https://doi.org/10.3389/fmolb.2023.1184200.

By regulating the pH of acidic organelles in eukaryotes, vacuolar V-type ATP-dependent proton pumps (V₁V_O) enable many critical cellular processes. They belong to a super-family of rotary ATPases and ATP synthases that include the F-type, A-type, and V/A-type. The V₁ complex consumes ATP, which drives rotation of its central rotor consisting of DFd subunits and the membrane-embedded c-ring of the V_O complex that pumps protons across the membrane. Cellular pH homeostasis is maintained in coordination with the metabolic state of the cell by reversible dissociation of V₁ from V_O, and subunit C from V₁, which disables ATP-driven proton pumping. Futile ATP hydrolysis of the dissociated V₁ is then inhibited by the regulatory subunit-H. The rotational dynamics of the eukaryotic S. cerevisiae V₁-ATPase lacking regulatory subunits H and C (V₁DHC) were characterized using single-molecule studies with high resolution of time and rotational position. Ensemble ATPase measurements showed that this motor had an apparent V_max at 1 mM Mg-ATP, which was subject to substrate inhibition at higher ATP concentrations. Rotation was observed in 120° power strokes separated by dwells comparable to the power strokes and catalytic dwells observed in F₁, A₁, and V/A₁ ATPases. However, V₁DHC rotation differed in that: (1) power stroke rotation often faltered during the first 60°; (2) an ATP-binding-like dwell often occurred 40° - 50° after the catalytic dwell, which did not increase in duration at limiting MgATP; (3) a dwell at ~115° after the catalytic dwell also was also observed; (4) limiting MgATP decreased power stroke angular velocity at rotary positions where dwells were not observed.

Non-tuberculous mycobacteria (NTM) infections are increasingly being reported worldwide. Intrinsic antibiotic resistance, impermeability of the cell wall to inhibitors, cleavage, modification of drugs within the pathogens and efficient efflux pumps are major obstacles to the treatment of NTM illness. The cure rate of NTM treatment is low, lengthy and costly, which in part is the result of low potency, poor clinical response, severe side-effects. To overcome these problems, new inhibitor targets, compounds and novel combinatory approaches are needed. As a strictly aerobic class of pathogens, NTM depend on the oxidative phosphorylation pathway to form ATP by F₁F_O-ATP synthase. Recent studies demonstrated that the NTM F₁F_O-ATP synthase is a potent target to inhibit growth of NTM by either binding to the F₁- [1-3] or F_O domain of this engine [4-5]. Here we present new and highly potent inhibitors with high efficacy against a broad spectrum of fast- and slow-growing NTMs including clinical isolates. They are mycobacterial-specific and non-toxic to the human gut biofilm or eukaryotic cells and enhance the potency of a variety of existing NTM drugs, thereby overcome the issues of drug tolerance and resistance. Finally, their chemical synthesis makes these molecules cost-effective and attractive inhibitors for pharma and healthcare.

[1] Ragunathan P., Dick, T., and Grüber, G. Anti-Mycobacterium abscessus activity of tuberculosis F-ATP synthase inhibitor GaMF1. Antimicrob. Agents and Chemother. 66 (2022):e0001822.

[2] Joon, S., Harikishore, A., Wong, C.-F., Ragunathan, P., Dick, T., and Grüber, G. Atomic solution structure of Mycobacterium abscessus F-ATP synthase subunit ε and identification of Ep1MabF1 as a targeted inhibitor. FEBS J. 289 (2022) 6308-6323.

[3] Wong, C.-F., Saw, W.-G., Basak, S., Sano, M., Ueno, H., Kerk, H. W., Litty, D., Ragunathan, P., Dick, T., Müller, V., Noji, H., and Grüber, G. Structural elements involved in ATP hydrolysis inhibition and ATP synthesis of tuberculosis and non-tuberculous mycobacterial F-ATP synthase decipher new targets for inhibitors. Antimicrobe. Agents and Chemother. 66 (2022) (12):e0105622.

[4] Sarathy JP, Ganapathy US, Zimmerman MD, Dartois V, Gengenbacher M, Dick T. TBAJ-876, a 3,5-Dialkoxypyridine Analogue of Bedaquiline, Is Active against Mycobacterium abscessus. Antimicrob Agents Chemother 64 (2020) e02404-19.

[5] Ragunathan, P. Sae-Lao, P., Hamela, C., Alcaraz, M., Krah, A. Poh, W.H., Pee, C.J.E., Lim, A.Y.H., Rice, S.A., Pethe, P., Bond, P.J., Dick, T., Kremer, L., Bates, R.W., and Grüber, G. High efficacy of the F-ATP synthase inhibitor TBAJ-5307 against non-tuberculous mycobacteria in vitro and in vivo. J. Biol. Chem. 300 (2024): 105618.

ATP synthases are the proteins that produce the energetic molecule in the cell through the rotatory movement of their membrane-spanning subunit linked to their catalytic head. In mitochondria, ATP synthases are found to arrange as dimers at high-curved edges of cristae. A direct link between the rotatory movement of ATP synthases and membrane structure has been suggested [1]. Here, we found an active curvature sorting of ATP synthases in lipid nanotubes pulled from giant vesicles. Coarse-grained simulations confirm the curvature-seeking behaviour of rotating ATP synthases, promoting reversible and frequent protein-protein contacts [2]. From simulations, we also observe that rotation of the transmembrane c-ring produces a hydrophobic mismatch that result on the formation of transient ATP synthase dimers. Protein dimerization relies on a weak membrane-mediated attractive interaction on the order of 1.5 k_BT. These transient dimers form a conic-like arrangement characterized by a wedge angle of 𝜽 ≈ 50°, producing a dynamic coupling between protein shape and membrane curvature. Our results suggest a new role of the rotational movement of ATP synthases for their dynamic self-assembly in bioenergetic membranes.

[1] V. Almendro-Vedia, et al., How rotating ATP synthases can modulate membrane structure, Archives of Biochemistry and Biophysics, 708 (2021), 108939.

[2] D. Valdivieso González, et al., Rotation of the c‐Ring Promotes the Curvature Sorting of Monomeric ATP Synthases, Advanced Science, 10 (2023), 2301606.

Mrp (multiple resistance and pH adaptation) type proton/sodium antiporters represent a unique group of multi-subunit cation/proton antiporters. They are closely related to the members of the complex I superfamily, with homologues of their proton translocating domain making up much of the core subunits of the membrane arm of respiratory complex I and homologues of their cation translocating domain being present in redox coupled sodium pumps like MBH and MBS.

In recent work, we have determined the cryo-EM structure of the Mrp antiporter from Bacillus pseudofirmus at 2.2 Å resolution and proposed that a histidine switch mechanism plays a crucial role in gated proton transfer [1]. The strictly conserved His248 in MrpA is thought to shuttle protons between pathways connecting to the periplasm, cytoplasm and to the sodium translocating domain. In this work, we present further insights into the inner workings of the histidine switch mechanism by means of structure guided mutagenesis of key residues. Our comprehensive study also includes residues whose mutation in complex I leads to mitochondrial disease.

[1] 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, Nat Commun 13 (2022) 6091. https://doi.org/10.1038/s41467-022-33640-y.

Voltage-Dependent Anion-selective Channel (VDAC) represents the most abundant pore- forming protein family of the outer mitochondrial membrane (OMM) and is the principal gatekeeper for ions and metabolites of the organelle. In humans, VDAC3 has 6 cysteine residues which can be in different redox states, while VDAC1 has only 2 cysteines [1]. Recently, our group suggested that cysteine residues of VDAC3, which are exposed to the intermembrane space (IMS), are indispensable for counteracting ROS-induced oxidative stress [2]. Mass spectrometry analysis revealed the ability of VDAC3 to form intra- and intermolecular disulfide bridges. This finding led us to test whether these disulfide bridges could have additional biological functions. To that aim, we investigate the role of cysteines residues in the biogenesis of VDAC3 and VDAC1. To monitor the biogenesis, in vitro import assays of radiolabeled 35S-VDAC3 and 35S-VDAC3-cysteineless were performed, while 35S- VDAC1 and 35S-VDAC1 cysteineless were used for comparison. The import kinetics of the radiolabeled proteins were investigated using mitochondria isolated from S. Cerevisiae cells. Since the Mia40 pathway facilitates disulfide bond formation of newly synthesized IMS proteins, we asked if the disulfide bridges of VDAC3 might be catalyzed by Mia40. Therefore, we compared import into control organelles to those isolated from MIA40 mutant strain. Our results show that VDAC3 biogenesis is not affected by mutating Mia40, and surprisingly the 35S-VDAC3 cysteineless variant is better imported into control mitochondria as compared to the native 35S-VDAC3. Moreover, in vitro import assays followed by BN- PAGE highlighted that cysteineless VDAC3 forms mature complexes faster than the native protein. Overall, our results suggest that the cysteine residues of VDAC3 slow down its biogenesis and maturation.

[1] S. Reina, M.G.G. Pittalà, F. Guarino, A. Messina, V. De Pinto, S. Foti, R. Saletti, Cysteine Oxidations in Mitochondrial Membrane Proteins: The Case of VDAC Isoforms in Mammals, Front Cell Dev Biol, 8 (2020) 397.
[2] S. Reina, S. Conti Nibali, MF. Tomasello, A. Magrì, A. Messina, V. De Pinto, Voltage Dependent Anion Channel 3 (VDAC3) protects mitochondria from oxidative stress, Redox Biol., 51 (2022) 102264.

Cation-proton antiporters (CPAs) are secondary-active transporters involved in the regulation of cellular cation, pH, and volume homeostasis. The bacterial NhaA antiporter belonging to the CPA2 family is an electrogenic transporter that exchanges a single Na+ for two H+ across the cell membrane. The activity of NhaA depends on pH, increasing by three orders of magnitude between pH 6.5 and pH 8.5. Despite extensive structural and functional characterizations, the exact mechanism of NhaA pH regulation remains unclear.
Here, we report constant-pH molecular dynamics results using a pH 8.5 cryo-EM structure, resolved in our laboratory. Our lambda-dynamics simulations recapitulate the experimentally observed increase in activity from pH 4.0 to 8.5. Na+ is ushered towards the active site by a pH sensor domain, where it interacts with active site residues to induce a pKa shift. In the absence of Na+, the active site residues maintain their charge, requiring significant pKa shifts. Over the pH range of 4.0 to 8.5, D133 remains deprotonated and thus negatively charged to compensate for the dipole between helices IVc and XIp. K300 remains positively charged (with a pKa not too different from the model value) to compensate for the IVp and XIc helix dipole. D163 is always protonated, so not to introduce excess negative charge into the protein interior. Even with D163 fully protonated, NhaA was open towards the periplasm, challenging the previously proposed role of D163 as the access-control residue. The deprotonation of D164 strongly correlates with the presence of Na+, in line with the concept of minimizing the extra charge within the transmembrane region. Separate simulations with Na+ excluded from the active site indicate a strong shift in the pKa of D164, and the residue is always protonated over the investigated pH range. This finding suggests that it is the Na+ ion that causes the deprotonation of D164.

Eukaryotic cell function and survival relies on the use of a mitochondrial H⁺ electrochemical gradient (Δp), which is composed by an inner mitochondrial membrane (IMM) potential (ΔΨmt) and a pH gradient (ΔpH). So far, ΔΨmt has been assumed to be composed exclusively by H⁺. Here, using a rainbow of mitochondrial and nuclear genetic models, we have discovered that a Na⁺ gradient equates with the H⁺ gradient and controls half of ΔΨmt in coupled respiring mammalian mitochondria. This parallelism is controlled by the activity of the long-sought Na⁺-specific Na⁺/H⁺ exchanger (mNHE), which we have identified as the P-module of complex I (CI). Deregulation of this mNHE function, without affecting its enzymatic activity or assembly of CI, occurs in Leber’s hereditary optic neuropathy (LHON), which has profound consequences in ΔΨmt and mitochondrial Ca²⁺ homeostasis and explains the previously unknown molecular pathogenesis of this neurodegenerative disease.

The mitochondrial oxidative phosphorylation system (OXPHOS) is pivotal for cellular energy production through ATP synthesis [1]. Despite its central role, the phosphate carrier (PiC) within OXPHOS has received limited attention. Here, we conducted in silico analysis on nuclear-expressed Drosophila melanogaster OXPHOS genes. Results revealed widespread expression, Nuclear Respiratory Gene (elements presence, and developmental-dependent expression, with distinct peaks during late embryonic and pupal stages. Additionally, we extensively characterized the D. melanogaster mitochondrial phosphate carrier (Mpcp). Two genes, CG9090 and CG4994, encode putative Mpcp isoforms (Mpcp1 and Mpcp2), exhibiting intriguingly different expression patterns during development compared to other OXPHOS genes. Both Mpcp isoforms are vital for ATP synthesis and organismal development, with CG9090 potentially influencing lifespan and aging processes. Functional assays, including complementation tests and swelling experiments in yeast mir1Δ strain, alongside kinetic characterization of recombinant mature Mpcp2, confirmed phosphate transport abilities. Notably, Mpcp1 displayed significantly lower activity than Mpcp2, suggesting potential functional differences akin to mammalian PiC-A and PiC-B [2]. Recently, extending the in silico analysis to all genes encoding mitochondrial carrier (MC) proteins revealed exclusive expression patterns for PiC and adenine nucleotide transporter (ANT), underscoring their integral roles in OXPHOS. This study emphasizes PiC's importance in ATP synthesis, organismal development, and aging. It reveals parallels between Drosophila and mammalian PiC isoforms, aiding OXPHOS disease research in mammals.

[1] R. Curcio, P. Lunetti, V. Zara, A. Ferramosca, F. Marra, G. Fiermonte, A.R. Cappello, F. De Leonardis, L. Capobianco, V. Dolce, Drosophila melanogaster mitochondrial carriers: similarities and differences with the human carriers, Int. J. Mol. Sci. 21 (17) (2020).

[2] Curcio R, Frattaruolo L, Marra F, Pesole G, Vozza A, Cappello AR, Fiorillo M, Lauria G, Ahmed A, Fiermonte G, Capobianco L, Dolce V. Two functionally different mitochondrial phosphate carriers support Drosophila melanogaster OXPHOS throughout distinct developmental stages. Biochim Biophys Acta Mol Cell Res. 2024

Yat Kei Lo^1,2, Michael Seletskiy², Stefan Bohn³, Adel Beghiah⁴, Ville Kaila⁴, Jan Schuller^1,2

¹Center for Synthetic Microbiology, University of Marburg, Marburg, Germany

²Department of Chemistry, University of Marburg, Marburg, Germany

³Helmholtz Center Munich, Munich, Germany

⁴Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden

jan.schuller@synmikro.uni-marburg.de

Unfolding the molecular mechanism of a novel membrane potential-coupled inorganic carbon-concentrating transporter

The dissolved inorganic carbon-concentrating transporter (DIC-CT) is a novel carbon-concentrating mechanism (CCM) that enables autotrophic bacteria to accumulate a high level of intracellular bicarbonate for sustaining carbon fixation [1]. This transporter was demonstrated to be crucial for chemoautotrophs [1,2], and can be found in more than 12 classes of bacteria, including several opportunistic pathogens [2,3]. Despite of its significant physiological role, the molecular mechanism of DIC-CT remains largely enigmatic. Here we present the first cryo-EM single particle analysis of a nanodisc embedded DIC-CT along with functional characterization. The system features a transmembrane subunit with notable similarities to the proton-pumping subunits of respiratory Complex I (NADH:ubiquinone oxidoreductase), and a cytoplasmic subunit that partly mimics carbonic anhydrases. We further identified key elements possibly involved in proton conduction, as well as catalysis of CO₂ hydration. Our findings suggest that the system might function as a unique class of proton motive force-coupled vectorial carbonic anhydrase, distinct from conventional cytoplasmic or carboxysomal carbonic anhydrases. This discovery not only provides mechanistic insights on bacterial CCM but also broadens our understanding on Complex I-like energy-coupled catalysis.

[1] Schmid S, et al. Dissolved Inorganic Carbon-Accumulating Complexes from Autotrophic Bacteria from Extreme Environments. J Bacteriol. 2021 Nov 5;203(23):e0037721

[2] Desmarais JJ, et al. DABs are inorganic carbon pumps found throughout prokaryotic phyla. Nat Microbiol. 2019 Dec;4(12):2204-2215

[3] Fan SH, et al. Role of the NaHCO₃ Transporter MpsABC in the NaHCO₃-β-Lactam-Responsive Phenotype in Methicillin-Resistant Staphylococcus aureus. Microbiol Spectr. 2023 Jun 15;11(3):e0014123.

As mitochondrial Ca²⁺ is crucial for metabolism and life/cell decision, regulation of its homeostasis is an important factor. Mitochondrial Ca²⁺ homeostasis is controlled by uptake and release transporters: mitochondrial Ca²⁺ uniporter complex (MCUC), Na⁺/Li⁺/Ca²⁺ exchanger (NCLX), and the Na⁺-independent Ca²⁺/H⁺ exchanger (CHE), which we molecularly identified as Transmembrane BAX inhibitor-1 motif-containing protein 5 (TMBIM5/MICS1)[1]. In addition, the mitochondrial permeability transition pore is a critical Ca²⁺ determinant through transient opening.

TMBIM5/MICS1 belongs to the evolutionary conserved TMBIM family, with TMBIM5 as the only mitochondrial member and BsYetJ as the bacterial ancestor. The crystal structure of BsYetJ suggests a pH-sensitive regulation of Ca²⁺ transport, which is likely mediated by highly conserved amino acid residues. Having previously demonstrated that TMBIM5 mediates mitochondrial Ca²⁺ and H⁺ fluxes in intact and permeabilised cells and in in-vitro systems, and that TMBIM5 interacts with LETM1 the mitochondrial K⁺/H⁺ exchanger (KHE)[1], now we are showing a mutational analysis exploring whether, similarly to the ancestral bacterial homolog BsYetJ model, TMBIM5 regulates Ca²⁺ fluxes by a pH-sensitive switch between an open and a closed conformation, and if the mutations affect the interaction with LETM1 and eventually it’s function. The amino acid sequence alignment between BsYetJ and TMBIM5 points to highly conserved residues proposed to regulate the conformational changes. We analysed the effect of mutating these residues on the protein function (CHE) and on the interaction with LETM1. Using a Ca²⁺ fluorophore and permeabilised cells, in which knockout of TMBIM5 (TMBIM5KO) abolishes CHE, we functionally explore the CHE activity in TMBIM5KO that re-express TMBIM5WT or TMBIM5 mutants. We then investigated the effect of these mutations on the interaction with LETM1 and eventually its role in mitochondrial KHE in the function of KOAc-induced mitochondrial swelling.

[1] S. Austin, R. Mekis, S.E.M. Mohammed, M. Scalise, W.-A. Wang, M. Galluccio, C. Pfeiffer, T. Borovec, K. Parapatics, D. Vitko, N. Dinhopl, N. Demaurex, K.L. Bennett, C. Indiveri, K. Nowikovsky, TMBIM5 is the Ca2+/H+ antiporter of mammalian mitochondria, EMBO Rep 23 (2022) e54978. https://doi.org/10.15252/EMBR.202254978.

Airways faces exposure to air pollution, which contributes to the onset of respiratory illnesses and fatalities. Urban dust, a major component of smog, contains particulate matter (PMs) that exhibit cytotoxic effects on human bronchial epithelial cells, including mitochondrial dysfunction. To counteract these effects, one potential approach is safeguarding mitochondria from PM-induced cytotoxicity, possibly by targeting mitochondrial potassium channels. Activation of these channels leads to a decrease in membrane potential, an increase of mitochondrial respiration, and a change in reactive oxygen species (ROS) levels. Pharmacological activation of mitochondrial K⁺ channels has shown promise in mitigating the negative effects of hypoxia/reperfusion injury in heart and brain tissue. Recently, we identified the large-conductance calcium-activated potassium (mitoBK_Ca) channels in the inner mitochondrial membrane of human bronchial epithelial cells. Our current research focuses on elucidating the role of mitoBK_Ca channels in mitochondrial physiology and their potential cytoprotective function against PM-induced cytotoxicity. In bronchial epithelium lacking a functional BK_Ca channel (HBE Δα), we observed impaired mitochondrial function. We observed a reduction in the maximum level of cellular respiration in HBE Δα cells compared to wild-type cells. Using Blue Native electrophoresis we also found that the lack of the BK_Ca channel causes changes in the respiratory chain complexes. Additionally, we assessed the expression of selected mitochondrial genes, including genes encoding the respiratory chain subunits. We also characterized the metabolic profile of bronchial cells when the BK_Ca channel was absent. In summary, our research demonstrates that the presence of the BK_Ca channel is important for the functioning of mitochondria in bronchial epithelial cells. This study was supported by a grant (2019/35/B/NZ1/02546) from the National Science Centre, Poland.

In order to meet the demands of a growing world population and improve crop yield it is necessary to understand the core reactions in photosynthesis. One of these is the water oxidation step, catalyzed by the manganese containing oxygen evolution complex in photosystem II (PSII). Manganese availability in the plant and thylakoid lumen may therefore be a key question for PSII activity. To study the effect of manganese concentration on photosynthesis, plant mutants with limited ion transport to the thylakoid can be used. One such mutant in Arabidopsis thaliana is pam71, which has been suggested to be a transport protein responsible for pumping manganese and/or calcium over the thylakoid membrane [1,2]. By comparing pam71 to the wildtype, information on growth, photosynthetic efficiency as well as the cellular organization under impaired ion transport can be retrieved. We have shown that the light response of photosynthesis differs between pam71 and wild type by measuring chlorophyll fluorescence kinetics. We have also applied super resolution fluorescence microscopy, which gives structural information on the thylakoid stacking in living leaf material. These results can be used to get a better understanding of the consequences of manganese and/or calcium deficiency on the photosynthetic electron transport and the organization of thylakoid membranes.

[1] A. Schneider, I. Steinberger, A. Herdean, C. Gandini, M. Eisenhut, S. Kurz, A. Morper, N. Hoecker, T. Rühle, M. Labs, U. Flügge, S. Geimer, S. Birkelund Schmidt, S. Husted, A. Weber, C. Spetea, D. Leister, The Evolutionarily Conserved Protein PHOTOSYNTHESIS AFFECTED MUTANT71 Is Required for Efficient Manganese Uptake at the Thylakoid Membrane in Arabidopsis, Plant Cell, 28 (2016) 892-910.

[2] J. Frank, R. Happeck, B. Meier, M. T. T. Hoang, J. Stribny, G. Hause, H. Ding, P. Morsomme, S. Baginsky, E. Peiter, Chloroplast-localized BICAT proteins shape stromal calcium signals and are required for efficient photosynthesis. New Phytol. 221 (2019) 866-880

In response to high Ca²⁺, the inner mitochondrial membrane can undergo a process defined as the permeability transition (PT), that leads to matrix swelling and eventually culminates in cell death initiation. The PT is due to the opening of the PT pore (PTP), a Ca²⁺-activated and high-conductance channel. Among PTP modulators, thiol reagents (e.g. phenylarsine oxide, PAO) are inducers, while acidic pH acts as inhibitor. The molecular nature of the PTP was highly debated. The adenine nucleotide translocator (ANT) was considered a key mediator, although genetic studies revealed that other components are likely involved. Recently, the ATP synthase was shown to generate Ca²⁺-dependent channels perfectly matching those of the PTP. Whether these two permeation pathways act independently responding to specific PT modulators or cooperate is not known. Here, we evaluated the relative contribution of ANT and ATP synthase in the PT, by analyzing the effect of ANT specific ligands bongkrekic acid (BKA) and atractylate (ATR), which inhibits and activates the ANT channel, respectively, on Ca²⁺-dependent swelling at different pH. At pH 7.4, BKA and ATR showed only partial effects on PT occurrence. Moreover, PAO does not induce the PT through the ANT as ANTs knock-out fibroblasts are still sensitive to the thiol reagent. Thus, at pH 7.4, ATP synthase likely predominates. Conversely, at pH 6.5 (a condition that prevents opening of the ATP synthase channel), the PT can be activated by ATR and fully prevented by BKA, suggesting a primary role of the ANT. Remarkably, at pH 6.5, benzodiazepine (Bz)-423, which binds OSCP subunit of the ATP synthase, triggers mitochondrial swelling that can be prevented by BKA, suggesting an intimate connection between ATP synthase and ANT. Consistently, co-immunoprecipitation experiments revealed an interaction between ANT and pore-forming subunits c and g of the ATP synthase, hinting at a possible cross-talk between the two candidates in the PT process.

The ABC transporter CydDC is conserved in most bacteria and plays a central role in the biogenesis of membrane-integrated and soluble cytochromes¹. However, the exact function and mechanisms of this heterodimeric bacterial ABC transporter remain unclear. In our study, we map the conformational landscape and resolve the so-far enigmatic function of the ubiquitous, medically relevant, but poorly understood bacterial ABC transporter CydDC. We capture this fascinating machine in action, as it powers heme transport for the assembly of redox enzymes.

In doing so, we used a combination of cellular, biochemical, structural and computational methods and established single-particle electron cryo-microscopy (cryo-EM) as a scalable analytical tool. We screened a large array of substrate candidates, sample conditions, and rationally designed mutant variants of CydDC. In total, we determined structures of CydDC in 23 different sample conditions at resolutions between 2.7 to 3.9 Å. This allowed us to unambiguously identify heme as the transport substrate of this transporter. On top of this, we were able to delineate the precise mechanism of substrate binding, gating, and transport from datasets obtained under biochemically defined and turnover inducted conditions. The membrane-accessible heme entry site of CydDC is primarily controlled by the conformational plasticity of CydD transmembrane helix 4, the extended cytoplasmic segment of which also couples heme confinement to a rotational movement of the CydC nucleotide-binding domain. Our cryo-EM data highlight that this signal transduction mechanism is necessary to drive conformational transitions toward occluded and outward-facing states. Atomistic molecular dynamics simulations were further employed to provide detailed insight into the conformational landscape of CydDC during substrate binding and occlusion. Our simulations reveal that heme binds laterally from the membrane space to the transmembrane region of CydDC, enabled by a highly asymmetrical inward-facing CydDC conformation. During the binding process, heme propionates interact with positively charged residues on the surface and later in the substrate-binding pocket of the transporter, causing the heme orientation to flip 180 degrees.

Our approach underscores that systematic structural biology is able to shed light on molecular processes of ABC transporters which remain elusive due to methodological limitations. Moreover, this work sets the stage for the development of novel antibacterial drugs and a new line of attack against M. tuberculosis and other pathogenic bacteria, where CydDC is critically important for respiratory re-wiring upon host infection.

The role of calcium in the stimulation of mitochondrial respiration in cells that mainly rely on oxidative phosphorylation for ATP production is well known [1]. However, in the Warburg effect, a metabolic phenotype characteristic of proliferating and tumor cells in which ATP is mainly produced by glycolysis, the functional relevance of calcium-dependent activation of mitochondrial function and its impact in proliferation are less evident.

First, we have investigated the pathways by which calcium modulates mitochondrial activity in different proliferating cells. We demonstrate that, contrary to initial views [2], the classical pathway for calcium-dependent activation of respiration by its import through the Mitochondrial Calcium Uniporter (MCU) is dispensable for respiration and proliferation. In contrast, we show that an alternative pathway accomplished by calcium-dependent mitochondrial aspartate/glutamate carriers (AGCs) is essential to sustain respiration and cytosolic NADH/NAD⁺ balance due to their participation in the malate-aspartate shuttle (MAS) of NADH. Secondly, cells lacking AGCs also show a strong proliferative defect, and we have studied the mechanism involved. Interestingly, we observe that aspartate supplementation is sufficient to restore proliferation without rescuing respiration defects or the cytosolic NADH/NAD⁺ balance. These results suggest that AGCs are required to sustain cytosolic aspartate levels for proliferation and that AGCs also function independently of their participation in MAS. These proliferative defects in AGCs KO cells could potentially lead to physiological defects in vivo, such as those described in mice lacking agc1/slc25a12 [3].

[1] A. Del Arco, L. González-Moreno, …, J. Satrústegui, Regulation of neuronal energy metabolism by calcium: Role of MCU and Aralar/malate-aspartate shuttle, BBA Mol. Cell Res, 1870 (2023) 119468

[2] C. Cárdenas, M. Müller, …, J. K. Foskett, Selective Vulnerability of Cancer Cells by Inhibition of Ca(2+) Transfer from Endoplasmic Reticulum to Mitochondria, Cell Rep, 14 (2016) 2313-2324

[3] M. A. Jalil, L. Begum, …,T. Saheki, Reduced N-acetylaspartate levels in mice lacking aralar, a brain- and muscle-type mitochondrial aspartate-glutamate carrier, JBC 280 (2005) 31333–31339

Mitoquinol is a derivative of CoQ10, designed to penetrate mitochondria via a highly charged triphenyl-phosphonium cation moiety. To date, mitochondria accumulation of oral Mitoquinol has not been demonstrated. In this study, Mitoquinol was shown to be preferentially transported and accumulated in the mitochondria (as exemplified by the liver) after oral dosing to mice.

Mitoquinol was detected in plasma, heart and liver at 30 min up to 6 hrs post-dose, where accumulation in the heart remained steady at 0.04µM from 2-6 hr. Mitochondrial accumulation in heart was below the limit of detection, while Mitoquinol in liver mitochondria had a longer half-life than Mitoquinol in whole liver. Plasma Mitoquinol 1 hr after dosing was 0.2 µM. In contrast, based on CoQ10 peak size, plasma CoQ10 concentration is estimated at only ~3 nM 1 hr after dosing. Chronic dosing of Mitoquinol showed accumulation in heart, being 7-fold higher after 1 week than with an acute dose. The average concentration of Mitoquinol in the liver mitochondria 2 h after a single dose was ~2.8 µmol/kg vs 4.2 µmol/kg with chronic dosing. Oral Mitoquinol can be detected in plasma heart and liver within 30 min and is degraded/excreted from the liver and plasma within 6 h of a single dosing but may accumulate in the heart with chronic dosing

Chronic inflammation increases risk of developing insulin resistance (IR) and type 2 diabetes (T2D). Some anti-inflammatory drugs are more effective in reducing this risk than others, suggesting that these drugs have favorable metabolic effects in addition to anti-inflammatory effects. One such drug could be diflunisal, difluorophenyl derivate of salicylate, which can activate AMP-activated protein kinase (AMPK), a major regulator of cellular metabolism and a promising pharmacological target for the treatment of IR and T2D. We investigated whether diflunisal can activate AMPK in and affect metabolism of skeletal muscle cells, which are one of the most important targets for the treatment of IR and T2D.

In rat skeletal muscle cells L6, diflunisal uncoupled mitochondria and activated AMPK in a dose-dependent manner and stimulated glucose uptake, glycolysis and oxidation of glucose and oleic acid. Increase in glucose uptake and catabolism of glucose and oleic acid is likely required to counteract less efficient mitochondrial production of ATP due to partial uncoupling of mitochondria. We observed several changes at the level of gene expression or protein phosphorylation that support observed metabolic changes: 1) increased expression of glucose transporter (GLUT) 1 mRNA (could lead to higher glucose uptake through GLUT1); 2) increased phosphorylation of Akt substrate of 160 kDa at Thr642 (could stimulate glucose uptake through GLUT4); 3) decreased phosphorylation of pyruvate dehydrogenase at Ser293 (could stimulate pyruvate dehydrogenase and thus oxidative metabolism of glucose); 4) increased phosphorylation of acetyl-CoA carboxylase at Ser79 (could stimulate oxidative metabolism of oleic acid).

In summary, our results show that diflunisal activates AMPK and stimulates uptake and catabolism of glucose in skeletal muscle cells, suggesting that treatment with diflunisal might improve systemic glucose homeostasis in individuals with inflammatory disorders and T2D.

Uncoupling protein 1 (UCP1) dissipates energy as heat in brown adipose tissue, making it a target for treating metabolic disorders. Here, we investigate how purine nucleotides inhibit respiration uncoupling by UCP1. Molecular dynamic simulations predict that GDP binds UCP1 close to the common substrate binding site of mitochondrial carriers with the base moiety interacting with conserved residues R92 and E191. We identify one helix-turn down in UCP1 cavity a triplet of uncharged residues, F88/I187/W281, forming hydrophobic contacts with nucleotides. To confirm our predictions, we performed high-resolution respiration assays in yeast spheroplast. Both I187A and W281A mutants increase the fatty acid­induced uncoupling activity of UCP1 and partially suppress the inhibition of UCP1 activity by nucleotides. The F88A/I187A/W281A triple mutant is overactivated by micromolar concentration of fatty acids even in the presence of millimolar concentrations of purine nucleotides. Decomposition of the triple mutation shows that I187 and W281, which are highly conserved among UCPs subfamily, carry the triplet phenotype. In simulations, E191 and W281 interact with purine but not pyrimidine bases. These results provide a molecular understanding of the selective inhibition of UCP1 by purine nucleotides.

Uncoupling protein 1 (UCP1) is a mitochondrial carrier that catalyses proton leak across the mitochondrial inner membrane to generate heat for thermoregulation. Inducing UCP1-dependent energy expenditure therapeutically is a potential avenue to treat metabolic disease, though how activating ligands such as fatty acids interact to activate proton leak by the protein is not well understood. The central cavity of UCP1 conserves a triplet of arginine residues (‘R-triplet’; R84, R183, R277, human UCP1 numbering), related by three-fold pseudosymmetry in the protein, equivalent to the substrate binding contact points in other mitochondrial carriers. These amino acid residues have been shown to be important for interacting with purine nucleotide inhibitors, along with other residues in the cavity, however, their potential role in interacting with fatty acid activators is unclear. Past studies did not find a loss of fatty acid activated proton leak by UCP1 containing single mutations of the R-triplet.

Here, through UCP1 expression in yeast, we have systematically generated and purified an array of UCP1 variants each containing double or triple mutations of the R-triplet to alanine or glutamine, as well as mutations at other key amino acids positions in the UCP1 central cavity. Through assessment of ligand-induced thermostabiliy shift analysis and UCP1 liposome proton leak assays, we find that, as expected, double and triple mutations of the R-triplet compromises purine nucleotide binding. However, in contrast to single mutations, these combination mutations generally lead to a reduction in fatty acid induced rates of proton leak, albeit with a retention of some activity depending on the severity of mutation. Our findings suggest that the ‘R-triplet’ is important for UCP1 function, similar to amino acids at these positions in other carriers, but where each arginine shares a common role and compensates to a degree for loss at either of the other positions.

COX6B belongs to the MT-CO3 module and has been implicated as an assembly subunit of complex 4 (cIV). This was corroborated by the presence of fully assembled cIV, albeit in reduced levels in patient cells harboring COX6B1 missense mutations. Unexpectedly, COX6B knock-out in HEK293 cells (6BKO) resulted in a profound decrease in cIV subunits, resembling the phenotype of early assembling subunit COX4 knock-out (4dKO). The major difference was the stabilization of the MT-CO1-COX4-COX5A assembly intermediate (MT-CO1 module) in 6BKO. Later intermediates containing part of the MT-CO2 module were present trace quantities detectable by complexome profiling. Several cIV subunits (COX4, MT-CO2, COX7A2, and COX7A2L) were associated with incomplete respirasome-like supercomplexes (SC I III₂IV_sub), whereas subassemblies of cIV monomer or dimer, lacking only COX6B were not detected. Neither 4dKO nor 6BKO could assemble functional cIV, which would consume oxygen. Expression of alternative oxidase (AOX) partially restored electron flow in 6BKO and allowed the COX assembly to proceed toward later stages. Notably, MT-CO2 showed an increase in steady-state levels and stability, as its signal could be detected at the 24-hour chase time point of metabolic labeling. Subunits from all three assembly modules were further stabilized in a later intermediate (IV_sub2) and in SC I III₂IV_sub in 6BKO AOX cells. Complexome profiling revealed that incomplete modules (MT-CO2 module lacking COX7B, COX7C, and COX8A; MT-CO3 module lacking COX6A1 and COX6B1) were assembled into cIV complex. Further characterization by COX IP/MS identified the presence of 11 cIV subunits in the resulting vestigial complex, with only COX6B1, COX6A, and NDUFA4 missing. This complex could only support a residual respiration rate despite the presence of a complete catalytic core, probably due to the disrupted cytochrome c binding. We conclude that AOX expression restores the redox conditions required for early cIV assembly and allows the characterization of the genuine cIV subcomplex formed without COX6B1. Interestingly, the testes-specific isoform COX6B2 did not assemble into cIV either in 6BKO or any other cell line tested, despite its successful targeting into the mitochondria.

This project was supported by the Czech Science Foundation (22-21082S).

The assembly sequence of mitochondrial complexes has been extensively studied in proliferating cells. These studies mostly reflect de novo assembly and provide limited information on the dynamics of protein complexes in differentiated cells and tissues. The state of protein complexes in post-mitotic tissues may be better understood as a balance between biosynthesis and degradation. An important question is whether protein complexes are always assembled de novo or whether remodelling and repair mechanisms maintain mitochondrial function. We combined complexome profiling and pulse stable isotope labelling of amino acids in cell culture (Pulsed-SILAC) to investigate the turnover and half-life of individual proteins within protein complexes in differentiated post-mitotic C2C12 myotubes. The results represent a comprehensive collection of data on the dynamics of all stable mitochondrial protein complexes. The complete turnover of all complexes of the oxidative phosphorylation system (OXPHOS) takes approximately one month. We identified subunits of complex I with higher turnover rates in specific regions of the electron transport module. NDUFA6 and NDUFA7 are super-turnover subunits with the highest exchange rates within OXPHOS proteins. Reduced turnover leads to mitochondrial disorders and reflects the importance of dynamic exchange and involved factors. We characterised service factors, including proteases and chaperones, in patients and mouse models that are involved in these quality control mechanisms and are essential to ensure full bioenergetic function in post-mitotic tissues.

Pancreatic beta cells are a key element of energy metabolism, as they secrete the hormone insulin. For the correct regulation of metabolism, it is necessary that the amount of secreted insulin corresponds to the level of glucose in the blood. The mitochondria of beta cells play a fundamental role in this adjustment and must produce ATP accurately in response to the level of incoming substrate. It is generally accepted that the rate of the respiratory chain and the overall function of mitochondria are significantly influenced by the architecture of the mitochondrial network as well as the interaction of mitochondria with other cellular organelles [1]. In this project, we, therefore, aimed to characterize the mitochondrial network and ultrastructure in primary pancreatic beta cells under insulin-secreting/non-insulin-secreting conditions. We used Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) and high-resolution Structured Illumination Microscopy (SIM) to monitor the changes. Visualization of mitochondria revealed significant changes in the arrangement of mitochondrial cristae as well as changes in the overall morphology of the mitochondrial network. Furthermore, we used volume electron microscopy data to quantify mitochondrial membrane contact sites with the endoplasmic reticulum and nuclear envelope. Segmentation of these organelles was done using the Empanada library [2], and the density and extent of membrane contact sites were evaluated using algorithms in the sci-kit image library for Python.

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

[1] I. Kawano, B. Bazila, P. Ježek, A. Dlasková, Mitochondrial Dynamics and Cristae Shape Changes During Metabolic Reprogramming, Antioxidants Redox Signal. 39 (2023) 684–707. https://doi.org/10.1089/ars.2023.0268.

[2] R. Conrad, K. Narayan, Instance segmentation of mitochondria in electron microscopy images with a generalist deep learning model trained on a diverse dataset, Cell Syst. 14 (2023) 58-71.e5. https://doi.org/10.1016/j.cels.2022.12.006.

Pancreatic β-cells sense glucose via the elevated ATP synthesis and redox signaling (mediated by NADPH-oxidase 4), providing glucose stimulated insulin secretion (GSIS) [1]. We observed that mitochondrial cristae became narrower in response to the enhanced ATP synthesis upon GSIS, hypothetically dependent on stabilization of rows of ATP-synthase dimers along the crista rims. To test this hypothesis, vestigial ATP-synthases lacking F_O-membrane subunit e or subunits e+g were studied in INS-1E cells, edited using CRISPR/Cas9. In control cells, focused-ion beam/scanning electron microscopy (FIB/SEM) tomography, transmission electron microscopy (TEM), and 3D superresolution microscopy evidenced the decreased intracristal volume with cristae narrowing upon transition from low glucose (3 mM, non-stimulating insulin release) to saturated glucose concentrations (>11 mM), stimulating GSIS. These changes ceased upon e and e+g ablation. Cells with ablated e and e+g possessed lower ATP-elevation responses to glucose and also GSIS kinetics was slightly and nearly completely impaired after e and e+g deletion, respectively. Tetramers, hexamers, and higher oligomers of the ATP-synthase were absent in BN-PAGE of the solubilized samples of mitochondria isolated from the cells containing vestigial ATP-synthases. These results reflect importance of subunits e and g for creation of sharp edges of cristae, determining their minimum orthogonal dimension (width) in TEM images and the thinnest crista lamellae in FIB/SEM images.

Supported by the GACR grant 21-01205S to P.J. and 23-05798S to H.E.

[1] L. Plecitá-Hlavatá, M. Jabůrek, B. Holendová, J. Tauber, V. Pavluch, Z. Berková, M. Cahová, K. Schröder, R.P. Brandes, D. Siemen, P. Ježek, Glucose-stimulated insulin secretion fundamentally requires H₂O₂ signaling by NADPH Oxidase 4, Diabetes, 69 (2020) 1341-1354.

The development and progression of cancer requires several adaptations, which enable and promote intense cell proliferation and decreased sensitivity to death signals. Cancer cells are known for their metabolic plasticity favouring intensified proliferation, survival and migration. Such plasticity also helps cancer cells to become resistant to chemotherapeutics. Metabolic adaptation of cancer cells often includes a shift from oxidative phosphorylation-based metabolism to glycolysis. Mitochondrial metabolism, as well as other key cellular processes like apoptosis are regulated by the crosstalk between mitochondria and ER.

We investigated changes in metabolism and proteome using a model of hepatocellular carcinoma (HCC) – HepG2 and HepG2/C3A cell lines. In contrast to HepG2, HepG2/C3A cell line after reaching confluency loses proliferative potential and stops producing cancer markers (e.g. alpha fetoprotein, AFP). This feature of HepG2/C3A allowed us to observe differences in metabolism between quiescent and proliferative phenotypes having distinct energy demands. Interestingly, quiescent HepG2/C3A has lower metabolic activity than proliferating HepG2 and HepG2/C3A cultures. We observed remarkable metabolic flexibility and adaptability of these HCC cell lines to modifications of culture conditions. Metabolic reprogramming in these cells is accompanied by the change in the levels of mitochondrial oxidative phosphorylation chain complexes subunits, TP53-induced glycolysis and apoptosis regulator (TIGAR), carboxylesterase 1 (CES1) and proteins localized in mitochondria – ER contact sites e.g. IP3R3. Additionally, the shift from proliferative to quiescent phenotype is accompanied by the reprogramming of glycolysis and mitochondrial bioenergetic pathways which is reflected in the sensitivity profiles of quiescent and proliferative HepG2/C3A cultures to anticancer compounds.

Our findings may help to point out metabolic pathways which are specifically affected during the oncogenic process and may in the future become a target to support chemotherapeutic interventions.

Acknowledgements: This work was funded by the National Science Centre (Poland) grants: UMO-2021/43/I/NZ3/00510) for M.L-A., P.J., B.P. and M.R.W. and UMO-2015/17/D/NZ1/00030 for M.L-A.

Mitochondria are organelles not only involved in cellular respiration but also in several other pathways important for cell life and death. They are not isolated within the cells but are closely interconnected with other organelles, among which the Endoplasmic Reticulum (ER). Defective ER-mitochondria crosstalk and ER stress impacts on several cellular functions as well as on important intracellular pathways that promote the cancer development. Modulation of ER-mitochondria contacts have a role in cancer development and resistance to pharmacological therapy by impacting on cellular bioenergetics and metabolism. More recently, we showed that a reduction of mitochondria-ER contacts sites, by downregulation of tethers, can tune cancer cells intracellular signaling (e.g. Wnt signaling) both in vitro and in vivo, ultimately impacting on cancer cells proliferation. In addition, we have demonstrated that organelle contacts are mutually regulated in response to metabolism rewiring so affecting cancer formation/progression and cancer cells sensitivity to drugs. These findings reveal that affecting mitochondria-ER tethering may be beneficial against cancer by altering the cellular signaling, and in turn sensitizing tumor cells to chemotherapeutic treatment.