Conference Agenda

The detailed molecular mechanism of the essential bioenergetic enzyme cytochrome bc₁ (or respiratory complex III) remains elusive. In the Q_o site, the bifurcated two-electron transfer proceeds from the quinol (Q) substrate to iron-sulfur (FeS) and heme b_L centers, but the two coupled proton transfers are less understood. Here we present classical molecular dynamics simulations, reaction profiles obtained with hybrid QM/MM potentials, and analysis with a semiclassical tunneling theory to probe possible proton acceptors and feasible transfer pathways in the Q_o site. We find this site is highly hydrated, and various proton wires may be formed transiently between the quinol substrate and conserved side- chains (His152, Tyr147, and Glu295 in R. sphaeroides numbering), with one to three intervening water molecules. These waters have low mobility and were experimentally resolved in recent structural models. The first quinol oxidation is a nonadiabatic electronic transfer to the FeS center coupled with proton tunneling to His152 [1]. For the second electron and proton transfer, we test different proton acceptors, including a recent proposal for Asp278. A Grotthuss proton-hopping mechanism is activated with feasible kinetics only in the one- electron-oxidized state (semiquinol). We also find that proton wires for the second transfer are redundant, explaining the results of mutational studies [2]. These results help to describe the Q-cycle of cytochrome bc₁ in detail and pave the way to understand the mechanism of proton-coupled electron bifurcation catalyzed by several bioenergetic enzymes.

[1] S. G. Camilo, F. Curtolo, V. V. Galasi, G. M. Arantes, Tunneling and nonadiabatic effects on a proton-coupled electron transfer model for the Q_o site in cytochrome bc₁, J. Chem. Inf. Model., 61 (2021), 1840-1849.

[2] S. G. Camilo, G. M. Arantes, Flexibility and Hydration of the Q_o Site Determine Multiple and Redundant Pathways for Redox-Coupled Proton Transfer in Cytochrome bc₁. (2024), pre-print at: https://eq5e.short.gy/bc1-pp .

The gasotransmitters hydrogen sulfide (H₂S) and nitric oxide (NO) play crucial signalling roles in many physiological processes and, in bacterial pathogens, they can confer resistance against oxidative stress, host immune responses and antibiotics [1]. Yet, they are long-known inhibitors of heme-copper terminal oxidases in the respiratory chain. The highly branched respiratory chain of Pseudomonas aeruginosa, an opportunistic pathogen causing life-threatening infections difficult to eradicate, includes four terminal oxidases of the heme-copper type (caa₃, cbb₃-1, cbb₃-2 and bo₃) and one oxidase of the bd-type, the cyanide-insensitive oxidase (CIO) [2]. One of the most distinctive and relevant features of cytochromes bd is to provide tolerance to noxious gases while playing their bioenergetic function [3]. As P. aeruginosa is exposed during infection to H₂S and NO, we tested the effect of these gaseous molecules on the O₂ reductase activity of its terminal oxidases performing oxygraphic measurements on membrane preparations from wild-type P. aeruginosa PAO1 and isogenic mutants depleted of either CIO or all the other terminal oxidases [4]. We provide evidence that respiration mediated by CIO is resistant to high levels of sulfide and that exogenous H₂S enhances CIO expression supporting bacterial growth. As expected, CIO is reversibly inhibited by NO, but after NO exhaustion activity recovery is full and fast, indicating a protective role of CIO under NO stress conditions. The impact of these findings on microbial pathophysiology is discussed.

[1] Pal, V.K.; Bandyopadhyay, P.; Singh, A. Hydrogen sulfide in physiology and pathogenesis of bacteria and viruses. IUBMB Life (2018) 70, 393–410.
[2] Arai, H.; Kawakami, T.; Osamura, T.; Hirai, T.; Sakai, Y.; Ishii, M. enzymatic characterization and in vivo function of five terminal oxidases in Pseudomonas aeruginosa. J Bacteriol (2014) 196, 4206– 4215, doi:10.1128/JB.02176-14.

[3] Borisov, V.B.; Siletsky, S.A.; Paiardini, A.; Hoogewijs, D.; Forte, E.; Giuffrè, A.; Poole, R.K. Bacterial oxidases of the cytochrome bd family: redox enzymes of unique structure, function, and utility as drug targets. Antioxid Redox Signal (2021), 34, 1280–1318
[4] Nastasi, Martina R., Caruso, L., Giordano, F., Mellini, M., Rampioni, G., Giuffrè, A., & Forte, E. Cyanide Insensitive Oxidase confers hydrogen sulfide and nitric oxide tolerance to Pseudomonas aeruginosa aerobic respiration. Antioxidants, (2024) 13(3), 383.

20 years ago, we introduced single-molecule FRET (smFRET) measurements to study subunit rotation and regulatory conformational changes in individual F_oF₁-ATP synthases in liposomes, either driven by ATP hydrolysis or during ATP synthesis [1]. However, observation times of freely diffusing proteoliposomes in a confocal microscope are limited by Brownian motion to tens of milliseconds. To counteract diffusive motion actively in real time, we have built a fast anti-Brownian electrokinetic trap (ABEL trap [2]) with a laser focus pattern and electrode feedback controlled by a FPGA. Using the ABEL trap for smFRET measurements we recorded fast subunit rotation in F_oF₁-ATP synthases at different ATP concentrations and revealed variable hydrolysis rates from enzyme to enzyme [3]. We showed how the ATP/ADP ratio affects the speed of ATP-driven ε-subunit rotation and the relative percentage of active F_oF₁ [4]. Here we present the substeps during ATP-driven γ-subunit rotation in individual F_oF₁-ATP synthases from E. coli.

To overcome the observation limits due to photobleaching of the FRET fluorophors, we develop a quantum sensing approach using a fluorescent Nitrogen-Vacancy (NV) center in a nanodiamond and a nanomagnet [5]. Both non-bleaching markers are attached to a single F_oF₁-ATP synthase. Distance and orientational changes of the local magnetic field will allow to monitor individual subunit rotations in solution for hundreds of seconds, and might enable the observation of single F_oF₁-ATP synthase at work during ATP synthesis. We present our progress in NV quantum sensing.

[1] M. Börsch, M. Diez, B. Zimmermann, R. Reuter, P. Gräber, Stepwise rotation of the γ-subunit of EF_OF₁-ATP synthase observed by intramolecular single-molecule fluorescence resonance energy transfer, FEBS lett, 527 (2002) 147-152.

[2] A. E. Cohen, W. E. Moerner, The Anti-Brownian ELectrophoretic Trap (ABEL Trap): Fabrication and Software, Proc SPIE, 5699 (2005) 293-305.

[3] T. Heitkamp, M. Börsch, Fast ATP-Dependent subunit rotation in reconstituted F_oF₁‑ATP synthase trapped in solution, J Phys Chem B, 125 (2021) 7638-7650.

[4] I. Pérez, T. Heitkamp, M. Börsch, Mechanism of ADP-Inhibited ATP Hydrolysis in Single Proton-Pumping F_oF₁-ATP Synthase Trapped in Solution, Int J Mol Sci, 24 (2023) 8442.

[5] I. Pérez, A. Krueger, J. Wrachtrup, F. Jelezko, M. Börsch, Single NV center in nanodiamond for quantum sensing of protein dynamics in an ABEL trap, Proc. SPIE, 12849 (2024) 1284906.

The mitochondrial permeability transition (mPT) is the main cause of necrotic and apoptotic cell death during degenerative diseases of the heart and brain. The opening of the mitochondrial permeability transition pore (mPTP) leads to the dissipation of the mitochondrial inner membrane potential, followed by the outer membrane rupture, and release of cytochrome c that triggers the apoptosis. Despite the vital importance of mPTP in controlling cell life and death pathways, its molecular identity, structure, and regulation are not fully understood. The growing evidence suggests the irreplaceable role of mitochondrial F₁F_o ATP synthase in forming the large conductance channel of mPTP. Here in this work, we used different compounds of therapeutic significance to assess their role in modulating the leak channel activity of ATP synthase. Purified ATP synthase from porcine heart mitochondria was used in planar lipid bilayer recordings and single-particle cryo-electron microscopy studies to find the structure-function relationship in the ATP synthase leak channel and its gating mechanism. These findings will unlock the development of structure-based therapeutic compounds targeting the ATP synthase c-subunit leak channel for treating mPTP-related diseases.

Animals that face environmental hypoxia throughout its life cycles, such as helminths and bivalves, possess specific biochemical adaptations crucial for their survival. Among these adaptations, one stands out: the utilization of an alternative electron transport chain (ETC). In this unique ETC, rhodoquinone (RQ), a benzoquinone closely related to ubiquinone (UQ), serves as the lipidic electron transporter, while fumarate, rather than oxygen, serves as the final electron acceptor. This unconventional ETC utilizes only complex I and II, with the latter operating in reverse compared to the conventional aerobic ETC, facilitated by the redox potential of RQ. Our recent work has uncovered how animals synthesize RQ. We showed that RQ biosynthesis requires 3-hydroxyanthranilic acid (3-HAA), which is derived from tryptophan through the kynurenine pathway. The prenylation of 3-HAA occurs through a COQ-2 isoform, which is unique to these lineages. After prenylation, the arylamine ring is further modified to form RQ using several enzymes common to the UQ biosynthetic pathway. These studies revealed that two distinct pathways for RQ biosynthesis have evolved independently in nature, since some bacteria and protists synthesize RQ from UQ. In addition to RQ biosynthesis studies, we are addressing the function of RQ-dependent ETC in animals, including its relevance for survival in sulfide rich environments. Finally, key steps in RQ biosynthesis and function that offer potential target to treat helminth infections will be discussed.