Mechanistic Principles of Hydrogen Evolution in the Membrane-Bound Hydrogenase.

The membrane-bound hydrogenase (Mbh) from Pyrococcus furiosus is an archaeal member of the Complex I superfamily. It catalyzes the reduction of protons to H2 gas powered by a [NiFe] active site and transduces the free energy into proton pumping and Na+/H+ exchange across the membrane. Despite recent structural advances, the mechanistic principles of H2 catalysis and ion transport in Mbh remain elusive. Here, we probe how the redox chemistry drives the reduction of the proton to H2 and how the catalysis couples to conformational dynamics in the membrane domain of Mbh. By combining large-scale quantum chemical density functional theory (DFT) and correlated ab initio wave function methods with atomistic molecular dynamics simulations, we show that the proton transfer reactions required for the catalysis are gated by electric field effects that direct the protons by water-mediated reactions from Glu21L toward the [NiFe] site, or alternatively along the nearby His75L pathway that also becomes energetically feasible in certain reaction steps. These local proton-coupled electron transfer (PCET) reactions induce conformational changes around the active site that provide a key coupling element via conserved loop structures to the ion transport activity. We find that H2 forms in a heterolytic proton reduction step, with spin crossovers tuning the energetics along key reaction steps. On a general level, our work showcases the role of electric fields in enzyme catalysis and how these effects are employed by the [NiFe] active site of Mbh to drive PCET reactions and ion transport.

Long-range charge transfer mechanism of the III2IV2 mycobacterial supercomplex.

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.

Proton Release Reactions in the Inward H+ Pump NsXeR.

Directional ion transport across biological membranes plays a central role in many cellular processes. Elucidating the molecular determinants for vectorial ion transport is key to understanding the functional mechanism of membrane-bound ion pumps. The extensive investigation of the light-driven proton pump bacteriorhodopsin from Halobacterium salinarum(HsBR) enabled a detailed description of outward proton transport. Although the structure of inward-directed proton pumping rhodopsins is very similar to HsBR, little is known about their protonation pathway, and hence, the molecular reasons for the vectoriality of proton translocation remain unclear. Here, we employ a combined experimental and theoretical approach to tracking protonation steps in the light-driven inward proton pump xenorhodopsin from Nanosalina sp. (NsXeR). Time-resolved infrared spectroscopy reveals the transient deprotonation of D220 concomitantly with deprotonation of the retinal Schiff base. Our molecular dynamics simulations support a proton release pathway from the retinal Schiff base via a hydrogen-bonded water wire leading to D220 that could provide a putative gating point for the proton release and with allosteric interactions to the retinal Schiff base. Our findings support the key role of D220 in mediating proton release to the cytoplasmic side and provide evidence that this residue is not the primary proton acceptor of the proton transiently released by the retinal Schiff base.

Quinone Catalysis Modulates Proton Transfer Reactions in the Membrane Domain of Respiratory Complex I.

Complex I is a redox-driven proton pump that drives electron transport chains and powers oxidative phosphorylation across all domains of life. Yet, despite recently resolved structures from multiple organisms, it still remains unclear how the redox reactions in Complex I trigger proton pumping up to 200 Å away from the active site. Here, we show that the proton-coupled electron transfer reactions during quinone reduction drive long-range conformational changes of conserved loops and trans-membrane (TM) helices in the membrane domain of Complex I from Yarrowia lipolytica. We find that the conformational switching triggers a π → α transition in a TM helix (TM3ND6) and establishes a proton pathway between the quinone chamber and the antiporter-like subunits, responsible for proton pumping. Our large-scale (>20 µs) atomistic molecular dynamics (MD) simulations in combination with quantum/classical (QM/MM) free energy calculations show that the helix transition controls the barrier for proton transfer reactions by wetting transitions and electrostatic effects. The conformational switching is enabled by re-arrangements of ion pairs that propagate from the quinone binding site to the membrane domain via an extended network of conserved residues. We find that these redox-driven changes create a conserved coupling network within the Complex I superfamily, with point mutations leading to drastic activity changes and mitochondrial disorders. On a general level, our findings illustrate how catalysis controls large-scale protein conformational changes and enables ion transport across biological membranes.

Molecular Basis of the Electron Bifurcation Mechanism in the [FeFe]-Hydrogenase Complex HydABC.

Electron bifurcation is a fundamental energy coupling mechanism widespread in microorganisms that thrive under anoxic conditions. These organisms employ hydrogen to reduce CO2, but the molecular mechanisms have remained enigmatic. The key enzyme responsible for powering these thermodynamically challenging reactions is the electron-bifurcating [FeFe]-hydrogenase HydABC that reduces low-potential ferredoxins (Fd) by oxidizing hydrogen gas (H2). By combining single-particle cryo-electron microscopy (cryoEM) under catalytic turnover conditions with site-directed mutagenesis experiments, functional studies, infrared spectroscopy, and molecular simulations, we show that HydABC from the acetogenic bacteria Acetobacterium woodii and Thermoanaerobacter kivui employ a single flavin mononucleotide (FMN) cofactor to establish electron transfer pathways to the NAD(P)+ and Fd reduction sites by a mechanism that is fundamentally different from classical flavin-based electron bifurcation enzymes. By modulation of the NAD(P)+ binding affinity via reduction of a nearby iron-sulfur cluster, HydABC switches between the exergonic NAD(P)+ reduction and endergonic Fd reduction modes. Our combined findings suggest that the conformational dynamics establish a redox-driven kinetic gate that prevents the backflow of the electrons from the Fd reduction branch toward the FMN site, providing a basis for understanding general mechanistic principles of electron-bifurcating hydrogenases.

Molecular Principles of Redox-Coupled Protonation Dynamics in Photosystem II.

Photosystem II (PSII) catalyzes light-driven water oxidization, releasing O2 into the atmosphere and transferring the electrons for the synthesis of biomass. However, despite decades of structural and functional studies, the water oxidation mechanism of PSII has remained puzzling and a major challenge for modern chemical research. Here, we show that PSII catalyzes redox-triggered proton transfer between its oxygen-evolving Mn4O5Ca cluster and a nearby cluster of conserved buried ion-pairs, which are connected to the bulk solvent via a proton pathway. By using multi-scale quantum and classical simulations, we find that oxidation of a redox-active Tyrz (Tyr161) lowers the reaction barrier for the water-mediated proton transfer from a Ca2+-bound water molecule (W3) to Asp61 via conformational changes in a nearby ion-pair (Asp61/Lys317). Deprotonation of this W3 substrate water triggers its migration toward Mn1 to a position identified in recent X-ray free-electron laser (XFEL) experiments [Ibrahim et al. Proc. Natl. Acad. Sci. USA 2020, 117, 12,624-12,635]. Further oxidation of the Mn4O5Ca cluster lowers the proton transfer barrier through the water ligand sphere of the Mn4O5Ca cluster to Asp61 via a similar ion-pair dissociation process, while the resulting Mn-bound oxo/oxyl species leads to O2 formation by a radical coupling mechanism. The proposed redox-coupled protonation mechanism shows a striking resemblance to functional motifs in other enzymes involved in biological energy conversion, with an interplay between hydration changes, ion-pair dynamics, and electric fields that modulate the catalytic barriers.

Bicarbonate-controlled reduction of oxygen by the QA semiquinone in Photosystem II in membranes.

Photosystem II (PSII), the water/plastoquinone photo-oxidoreductase, plays a key energy input role in the biosphere. [Formula: see text], the reduced semiquinone form of the nonexchangeable quinone, is often considered capable of a side reaction with O2, forming superoxide, but this reaction has not yet been demonstrated experimentally. Here, using chlorophyll fluorescence in plant PSII membranes, we show that O2 does oxidize [Formula: see text] at physiological O2 concentrations with a t1/2 of 10 s. Superoxide is formed stoichiometrically, and the reaction kinetics are controlled by the accessibility of O2 to a binding site near [Formula: see text], with an apparent dissociation constant of 70 ± 20 µM. Unexpectedly, [Formula: see text] could only reduce O2 when bicarbonate was absent from its binding site on the nonheme iron (Fe2+) and the addition of bicarbonate or formate blocked the O2-dependant decay of [Formula: see text] These results, together with molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics calculations, indicate that electron transfer from [Formula: see text] to O2 occurs when the O2 is bound to the empty bicarbonate site on Fe2+ A protective role for bicarbonate in PSII was recently reported, involving long-lived [Formula: see text] triggering bicarbonate dissociation from Fe2+ [Brinkert et al, Proc. Natl. Acad. Sci. U.S.A. 113, 12144-12149 (2016)]. The present findings extend this mechanism by showing that bicarbonate release allows O2 to bind to Fe2+ and to oxidize [Formula: see text] This could be beneficial by oxidizing [Formula: see text] and by producing superoxide, a chemical signal for the overreduced state of the electron transfer chain.

Functional Dynamics of an Ancient Membrane-Bound Hydrogenase.

The membrane-bound hydrogenase (Mbh) is a redox-driven Na+/H+ transporter that employs the energy from hydrogen gas (H2) production to catalyze proton pumping and Na+/H+ exchange across cytoplasmic membranes of archaea. Despite a recently resolved structure of this ancient energy-transducing enzyme [Yu et al. Cell 2018, 173, 1636-1649], the molecular principles of its redox-driven ion-transport mechanism remain puzzling and of major interest for understanding bioenergetic principles of early cells. Here we use atomistic molecular dynamics (MD) simulations in combination with data clustering methods and quantum chemical calculations to probe principles underlying proton reduction as well as proton and sodium transport in Mbh from the hyperthermophilic archaeon Pyrococcus furiosus. We identify putative Na+ binding sites and proton pathways leading across the membrane and to the NiFe-active center as well as conformational changes that regulate ion uptake. We suggest that Na+ binding and protonation changes at a putative ion-binding site couple to proton transfer across the antiporter-like MbhH subunit by modulating the conformational state of a conserved ion pair at the subunit interface. Our findings illustrate conserved coupling principles within the complex I superfamily and provide functional insight into archaeal energy transduction mechanisms.

Deactivation blocks proton pathways in the mitochondrial complex I.

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.

Design of buried charged networks in artificial proteins.

Soluble proteins are universally packed with a hydrophobic core and a polar surface that drive the protein folding process. Yet charged networks within the central protein core are often indispensable for the biological function. Here, we show that natural buried ion-pairs are stabilised by amphiphilic residues that electrostatically shield the charged motif from its surroundings to gain structural stability. To explore this effect, we build artificial proteins with buried ion-pairs by combining directed computational design and biophysical experiments. Our findings illustrate how perturbation in charged networks can introduce structural rearrangements to compensate for desolvation effects. We validate the physical principles by resolving high-resolution atomic structures of the artificial proteins that are resistant towards unfolding at extreme temperatures and harsh chemical conditions. Our findings provide a molecular understanding of functional charged networks and how point mutations may alter the protein's conformational landscape.

Structure of inhibitor-bound mammalian complex I.

Respiratory complex I (NADH:ubiquinone oxidoreductase) captures the free energy from oxidising NADH and reducing ubiquinone to drive protons across the mitochondrial inner membrane and power oxidative phosphorylation. Recent cryo-EM analyses have produced near-complete models of the mammalian complex, but leave the molecular principles of its long-range energy coupling mechanism open to debate. Here, we describe the 3.0-Å resolution cryo-EM structure of complex I from mouse heart mitochondria with a substrate-like inhibitor, piericidin A, bound in the ubiquinone-binding active site. We combine our structural analyses with both functional and computational studies to demonstrate competitive inhibitor binding poses and provide evidence that two inhibitor molecules bind end-to-end in the long substrate binding channel. Our findings reveal information about the mechanisms of inhibition and substrate reduction that are central for understanding the principles of energy transduction in mammalian complex I.

Conformational dynamics modulate the catalytic activity of the molecular chaperone Hsp90.

The heat shock protein 90 (Hsp90) is a molecular chaperone that employs the free energy of ATP hydrolysis to control the folding and activation of several client proteins in the eukaryotic cell. To elucidate how the local ATPase reaction in the active site couples to the global conformational dynamics of Hsp90, we integrate here large-scale molecular simulations with biophysical experiments. We show that the conformational switching of conserved ion pairs between the N-terminal domain, harbouring the active site, and the middle domain strongly modulates the catalytic barrier of the ATP-hydrolysis reaction by electrostatic forces. Our combined findings provide a mechanistic model for the coupling between catalysis and protein dynamics in Hsp90, and show how long-range coupling effects can modulate enzymatic activity.

Redox-coupled proton pumping drives carbon concentration in the photosynthetic complex I.

Photosynthetic organisms capture light energy to drive their energy metabolism, and employ the chemical reducing power to convert carbon dioxide (CO2) into organic molecules. Photorespiration, however, significantly reduces the photosynthetic yields. To survive under low CO2 concentrations, cyanobacteria evolved unique carbon-concentration mechanisms that enhance the efficiency of photosynthetic CO2 fixation, for which the molecular principles have remained unknown. We show here how modular adaptations enabled the cyanobacterial photosynthetic complex I to concentrate CO2 using a redox-driven proton-pumping machinery. Our cryo-electron microscopy structure at 3.2 Å resolution shows a catalytic carbonic anhydrase module that harbours a Zn2+ active site, with connectivity to proton-pumping subunits that are activated by electron transfer from photosystem I. Our findings illustrate molecular principles in the photosynthetic complex I machinery that enabled cyanobacteria to survive in drastically changing CO2 conditions.

Ion Binding and Selectivity of the Na+/H+ Antiporter MjNhaP1 from Experiment and Simulation.

Cells employ membrane-embedded antiporter proteins to control their pH, salt concentration, and volume. The large family of cation/proton antiporters is dominated by Na+/H+ antiporters that exchange sodium ions against protons, but homologous K+/H+ exchangers have recently been characterized. We show experimentally that the electroneutral antiporter NhaP1 of Methanocaldococcus jannaschii (MjNhaP1) is highly selective for Na+ ions. We then characterize the ion selectivity in both the inward-open and outward-open states of MjNhaP1 using classical molecular dynamics simulations, free energy calculations, and hybrid quantum/classical (QM/MM) simulations. We show that MjNhaP1 is highly selective for binding of Na+ over K+ in the inward-open state, yet it is only weakly selective in the outward-open state. These findings are consistent with the function of MjNhaP1 as a sodium-driven deacidifier of the cytosol that maintains a high cytosolic K+ concentration in environments of high salinity. By combining experiment and computation, we gain mechanistic insight into the Na+/H+ transport mechanism and help elucidate the molecular basis for ion selectivity in cation/proton exchangers.

Quantum Chemical and QM/MM Models in Biochemistry.

Quantum chemical (QC) calculations provide a basis for deriving a microscopic understanding of enzymes and photobiological systems. Here we describe how QC models can be used to explore the electronic structure, dynamics, and energetics of biomolecules. We introduce the hybrid quantum mechanics/classical mechanics (QM/MM) approach, where a quantum mechanically described system of interest is embedded in a classically described force field representation of the biochemical surroundings. We also discuss the QM cluster model approach, as well as embedding theories, that provide complementary methodologies to model quantum mechanical effects in biomolecules. The chapter also provides some practical guides for building quantum biochemical models using the quinone reduction catalysis in respiratory complex I and a model reaction in solution as examples.

Electric field modulated redox-driven protonation and hydration energetics in energy converting enzymes.

Biological energy conversion is catalysed by proton-coupled electron transfer (PCET) reactions that form the chemical basis of respiratory and photosynthetic enzymes. Despite recent advances in structural, biophysical, and computational experiments, the mechanistic principles of these reactions still remain elusive. Based on common functional features observed in redox enzymes, we study here generic mechanistic models for water-mediated long-range PCET reactions. We show how a redox reaction within a buried protein environment creates an electric field that induces hydration changes between the proton acceptor and donor groups, and in turn, lowers the reaction barrier and increases the thermodynamic driving forces for the water-mediated PCET process. We predict linear free energy relationships, and discuss the proposed mechanism in context of PCET in cytochrome c oxidase.

Redox-coupled quinone dynamics in the respiratory complex I.

Complex I couples the free energy released from quinone (Q) reduction to pump protons across the biological membrane in the respiratory chains of mitochondria and many bacteria. The Q reduction site is separated by a large distance from the proton-pumping membrane domain. To address the molecular mechanism of this long-range proton-electron coupling, we perform here full atomistic molecular dynamics simulations, free energy calculations, and continuum electrostatics calculations on complex I from Thermus thermophilus We show that the dynamics of Q is redox-state-dependent, and that quinol, QH2, moves out of its reduction site and into a site in the Q tunnel that is occupied by a Q analog in a crystal structure of Yarrowia lipolytica We also identify a second Q-binding site near the opening of the Q tunnel in the membrane domain, where the Q headgroup forms strong interactions with a cluster of aromatic and charged residues, while the Q tail resides in the lipid membrane. We estimate the effective diffusion coefficient of Q in the tunnel, and in turn the characteristic time for Q to reach the active site and for QH2 to escape to the membrane. Our simulations show that Q moves along the Q tunnel in a redox-state-dependent manner, with distinct binding sites formed by conserved residue clusters. The motion of Q to these binding sites is proposed to be coupled to the proton-pumping machinery in complex I.

Terminal Electron-Proton Transfer Dynamics in the Quinone Reduction of Respiratory Complex I.

Complex I functions as a redox-driven proton pump in aerobic respiratory chains. By reducing quinone (Q), complex I employs the free energy released in the process to thermodynamically drive proton pumping across its membrane domain. The initial Q reduction step plays a central role in activating the proton pumping machinery. In order to probe the energetics, dynamics, and molecular mechanism for the proton-coupled electron transfer process linked to the Q reduction, we employ here multiscale quantum and classical molecular simulations. We identify that both ubiquinone (UQ) and menaquinone (MQ) can form stacking and hydrogen-bonded interactions with the conserved Q-binding-site residue His-38 and that conformational changes between these binding modes modulate the Q redox potentials and the rate of electron transfer (eT) from the terminal N2 iron-sulfur center. We further observe that, while the transient formation of semiquinone is not proton-coupled, the second eT process couples to a semiconcerted proton uptake from conserved tyrosine (Tyr-87) and histidine (His-38) residues within the active site. Our calculations indicate that both UQ and MQ have low redox potentials around -260 and -230 mV, respectively, in the Q-binding site, respectively, suggesting that release of the Q toward the membrane is coupled to an energy transduction step that could thermodynamically drive proton pumping in complex I.

Symmetry-related proton transfer pathways in respiratory complex I.

Complex I functions as the initial electron acceptor in aerobic respiratory chains of most organisms. This gigantic redox-driven enzyme employs the energy from quinone reduction to pump protons across its complete approximately 200-Å membrane domain, thermodynamically driving synthesis of ATP. Despite recently resolved structures from several species, the molecular mechanism by which complex I catalyzes this long-range proton-coupled electron transfer process, however, still remains unclear. We perform here large-scale classical and quantum molecular simulations to study the function of the proton pump in complex I from Thermus thermophilus The simulations suggest that proton channels are established at symmetry-related locations in four subunits of the membrane domain. The channels open up by formation of quasi one-dimensional water chains that are sensitive to the protonation states of buried residues at structurally conserved broken helix elements. Our combined data provide mechanistic insight into long-range coupling effects and predictions for site-directed mutagenesis experiments.

Energetics and dynamics of a light-driven sodium-pumping rhodopsin.

The conversion of light energy into ion gradients across biological membranes is one of the most fundamental reactions in primary biological energy transduction. Recently, the structure of the first light-activated Na+ pump, Krokinobacter eikastus rhodopsin 2 (KR2), was resolved at atomic resolution [Kato HE, et al. (2015) Nature 521:48-53]. To elucidate its molecular mechanism for Na+ pumping, we perform here extensive classical and quantum molecular dynamics (MD) simulations of transient photocycle states. Our simulations show how the dynamics of key residues regulate water and ion access between the bulk and the buried light-triggered retinal site. We identify putative Na+ binding sites and show how protonation and conformational changes gate the ion through these sites toward the extracellular side. We further show by correlated ab initio quantum chemical calculations that the obtained putative photocycle intermediates are in close agreement with experimental transient optical spectroscopic data. The combined results of the ion translocation and gating mechanisms in KR2 may provide a basis for the rational design of novel light-driven ion pumps with optogenetic applications.