Reactive oxygen species (ROS) are physiologically harmful radical species generated as byproducts of aerobic respiration. To detoxify ROS, most cells employ superoxide scavenging enzymes that disproportionate superoxide (O₂^·–) to oxygen (O₂) and hydrogen peroxide (H₂O₂). In contrast, the membrane-bound superoxide oxidase (SOO) is a minimal 4-helical bundle protein that catalyzes the direct oxidation of O₂^·– to O₂ and drives quinone reduction by mechanistic principles that remain unknown. Here, we combine multiscale hybrid quantum/classical (QM/MM) free energy calculations and microsecond molecular dynamics simulations with functional assays and site-directed mutagenesis experiments to probe the mechanistic principles underlying the charge transfer reactions of the superoxide-driven quinone reduction. We characterize a cluster of charged residues at the periplasmic side of the membrane that functions as a O₂^·– collecting antenna, initiating electron transfer via two b hemes to the active site for quinone reduction at the cytoplasmic side. Based on multidimensional QM/MM string simulations, we find that a proton-coupled electron transfer (PCET) reaction from the active site heme b and nearby histidine residues (H87, H158) results in quinol (QH₂) formation, followed by proton uptake from the cytoplasmic side of the membrane. The functional relevance of the identified residues is supported by site-directed mutagenesis and activity assays, with mutations leading to inhibition of the O₂^·–-driven quinone reduction activity. We suggest that the charge transfer reactions could build up a proton motive force that supports the bacterial energy transduction machinery, while the PCET machinery provides unique design principles of a minimal oxidoreductase.

Aerobic life is powered by membrane-bound redox enzymes that shuttle electrons to oxygen and transfer protons across a biological membrane. Structural studies suggest that these energy-transducing enzymes operate as higher-order supercomplexes, but their functional role remains poorly understood and highly debated. Here we resolve the functional dynamics of the 0.7 MDa III₂IV₂ obligate supercomplex from Mycobacterium smegmatis, a close relative of M. tuberculosis, the causative agent of tuberculosis. By combining computational, biochemical, and high-resolution (2.3 Å) cryo-electron microscopy experiments, we show how the mycobacterial supercomplex catalyses long-range charge transport from its menaquinol oxidation site to the binuclear active site for oxygen reduction. Our data reveal proton and electron pathways responsible for the charge transfer reactions, mechanistic principles of the quinone catalysis, and how unique molecular adaptations, water molecules, and lipid interactions enable the proton-coupled electron transfer (PCET) reactions. Our combined findings provide a mechanistic blueprint of mycobacterial supercomplexes and a basis for developing drugs against pathogenic bacteria.

Life is a non-equilibrium state and maintaining it thus requires a constant supply of external energy. To this end, organisms consume nutrients and convert the chemical energy into the universal energy carrier ATP. The energy conversion is achieved by the respiratory chain, which comprises multiple membrane-bound enzymes that convert the chemical energy into an electrochemical proton gradient, which is stored across a biological membrane. This proton gradient is, in turn, consumed by ATP synthase to produce ATP. On a molecular level, this is realized by a series of charge transfer processes, which are catalyzed by the respiratory chain complexes I-IV. These enzymes can also combine into larger assemblies, so-called supercomplexes, although their functional role remains highly debated.

In this thesis I will discuss the function of complexes I, III, and IV as well as the mycobacterial III₂IV₂ obligate supercomplex and the superoxide scavenger superoxide oxidase. To elucidate key functional aspects of these enzymes we have employed computational methods together with structural data and experimental measurements. Specifically, we have investigated the mechanism of complex I deactivation, as well as the proton transfer mechanics in its membrane domain. In complex IV, we have identified a mechanism by which steroid molecules can inhibit the enzyme, and describe how electric fields can selectively direct protons along specific pathways. Moreover, we have explored long-range charge transfer mechanisms in the unique mycobacterial III₂IV₂ supercomplex, and investigated the mechanism of the unique, membrane-bound superoxide scavenger superoxide oxidase. The combined results shed light on the molecular mechanisms that enable these enzymes to transduce energy.

Aerobic life is powered by membrane-bound enzymes that catalyze the transfer of electrons to oxygen and protons across a biological membrane. Cytochrome c oxidase (CcO) functions as a terminal electron acceptor in mitochondrial and bacterial respiratory chains, driving cellular respiration and transducing the free energy from O₂ reduction into proton pumping. Here we show that CcO creates orientated electric fields around a nonpolar cavity next to the active site, establishing a molecular switch that directs the protons along distinct pathways. By combining large-scale quantum chemical density functional theory (DFT) calculations with hybrid quantum mechanics/ molecular mechanics (QM/MM) simulations and atomistic molecular dynamics (MD) explorations, we find that reduction of the electron donor, heme a, leads to dissociation of an arginine (Arg438)–heme a₃ D-propionate ion-pair. This ion-pair dissociation creates a strong electric field of up to 1 V Å^-1 along a water-mediated proton array leading to a transient proton loading site (PLS) near the active site. Protonation of the PLS triggers the reduction of the active site, which in turn aligns the electric field vectors along a second, “chemical,” proton pathway. We find a linear energy relationship of the proton transfer barrier with the electric field strength that explains the effectivity of the gating process. Our mechanism shows distinct similarities to principles also found in other energy-converting enzymes, suggesting that orientated electric fields generally control enzyme catalysis.

The mitochondrial electron transport chain maintains the proton motive force that powers adenosine triphosphate (ATP) synthesis. The energy for this process comes from oxidation of reduced nicotinamide adenine dinucleotide (NADH) and succinate, with the electrons from this oxidation passed via intermediate carriers to oxygen. Complex IV (CIV), the terminal oxidase, transfers electrons from the intermediate electron carrier cytochrome c to oxygen, contributing to the proton motive force in the process. Within CIV, protons move through the K and D pathways during turnover. The former is responsible for transferring two protons to the enzyme’s catalytic site upon its reduction, where they eventually combine with oxygen and electrons to form water. CIV is the main site for respiratory regulation, and although previous studies showed that steroid binding can regulate CIV activity, little is known about how this regulation occurs. Here, we characterize the interaction between CIV and steroids using a combination of kinetic experiments, structure determination, and molecular simulations. We show that molecules with a sterol moiety, such as glyco-diosgenin and cholesteryl hemisuccinate, reversibly inhibit CIV. Flash photolysis experiments probing the rapid equilibration of electrons within CIV demonstrate that binding of these molecules inhibits proton uptake through the K pathway. Single particle cryogenic electron microscopy (cryo-EM) of CIV with glyco-diosgenin reveals a previously undescribed steroid binding site adjacent to the K pathway, and molecular simulations suggest that the steroid binding modulates the conformational dynamics of key residues and proton transfer kinetics within this pathway. The binding pose of the sterol group sheds light on possible structural gating mechanisms in the CIV catalytic cycle.

Cellular respiration is powered by membrane-bound redox enzymes that convert chemical energy into an electrochemical proton gradient and drive the energy metabolism. By combining large-scale classical and quantum mechanical simulations with cryo-electron microscopy data, we resolve here molecular details of conformational changes linked to proton pumping in the mammalian complex I. Our data suggest that complex I deactivation blocks water-mediated proton transfer between a membrane bound quinone site and proton-pumping modules, decoupling the energy-transduction machinery. We identify a putative gating region at the interface between membrane domain subunits ND1 and ND3/ND4L/ND6 that modulates the proton transfer by conformational changes in transmembrane helices and bulky residues. The region is perturbed by mutations linked to human mitochondrial disorders and is suggested to also undergo conformational changes during catalysis of simpler complex I variants that lack the "active"-to-"deactive" transition. Our findings suggest that conformational changes in transmembrane helices modulate the proton transfer dynamics by wetting/dewetting transitions and provide important functional insight into the mammalian respiratory complex I.

The respiratory complex I is a gigantic (1 MDa) redox-driven proton pump that reduces the ubiquinone pool and generates proton motive force to power ATP synthesis in mitochondria. Despite resolved molecular structures and biochemical characterization of the enzyme from multiple organisms, its long-range (similar to 300 A) proton-coupled electron transfer (PCET) mechanism remains unsolved. We employ here microsecond molecular dynamics simulations to probe the dynamics of the mammalian complex I in combination with hybrid quantum/classical (QM/MM) free energy calculations to explore how proton pumping reactions are triggered within its 200 A wide membrane domain. Our simulations predict extensive hydration dynamics of the antiporter-like subunits in complex I that enable lateral proton transfer reactions on a microsecond time scale. We further show how the coupling between conserved ion pairs and charged residues modulate the proton transfer dynamics, and how transmembrane helices and gating residues control the hydration process. Our findings suggest that the mammalian complex I pumps protons by tightly linked conformational and electrostatic coupling principles.