Photosystem II (PSII) is powered by the light-capturing properties of chlorophyll a pigments that define the spectral range of oxygenic photosynthesis. Some photosynthetic cyanobacteria can acclimate to growth in longer wavelength light by replacing five chlorophylls with long wavelength pigments in specific locations, including one in the reaction center (RC) (Science, 2018, 360, 1210-1213). However, the exact location and the nature of these long wavelength pigments still remain uncertain. Here we have addressed the color-tuning mechanism of the far-red light PSII (FRL-PSII) by excited state calculations at both the ab initio correlated (ADC2) and linear-response time-dependent density functional theory (LR-TDDFT) levels in combination with large-scale hybrid quantum/classical (QM/MM) simulations and atomistic molecular dynamics. We show that substitution of a single chlorophyll pigment (ChlD1) at the RC by chlorophyll d leads to a spectral shift beyond the far-red light limit, as a result of the protein electrostatic, polarization and electronic coupling effects that reproduce key structural and spectroscopic observations. Pigment substitution at the ChlD1 site further results in a low site energy within the RC that could function as a sink for the excitation energy and initiate the primary charge separation reaction, driving the water oxidation. Our findings provide a basis for understanding color-tuning mechanisms and bioenergetic principles of oxygenic photosynthesis at the far-red light limit.

The respiratory Complex I is a highly intricate redox-driven proton pump that powers oxidative phosphorylation across all domains of life. Yet, despite major efforts in recent decades, its long-range energy transduction principles remain highly debated. We create here minimal proton-conducting membrane modules by engineering and dissecting the key elements of the bacterial Complex I. By combining biophysical, biochemical, and computational experiments, we show that the isolated antiporter-like modules of Complex I comprise all functional elements required for conducting protons across proteoliposome membranes. We find that the rate of proton conduction is controlled by conformational changes of buried ion-pairs that modulate the reaction barriers by electric field effects. The proton conduction is also modulated by bulky residues along the proton channels that are key for establishing a tightly coupled proton pumping machinery in Complex I. Our findings provide direct experimental evidence that the individual antiporter modules are responsible for the proton transport activity of Complex I. On a general level, our findings highlight electrostatic and conformational coupling mechanisms in the modular energy-transduction machinery of Complex I with distinct similarities to other enzymes.

Tuberculosis is one of the most common causes of death worldwide, with a rapid emergence of multi-drug-resistant strains underscoring the need for new antituberculosis drugs. Recent studies indicate that lansoprazole—a known gastric proton pump inhibitor and its intracellular metabolite, lansoprazole sulfide (LPZS)—are potential antituberculosis compounds. Yet, their inhibitory mechanism and site of action still remain unknown. Here, we combine biochemical, computational, and structural approaches to probe the interaction of LPZS with the respiratory chain supercomplex III₂IV₂ of Mycobacterium smegmatis, a close homolog of Mycobacterium tuberculosis supercomplex. We show that LPZS binds to the Q_o cavity of the mycobacterial supercomplex, inhibiting the quinol substrate oxidation process and the activity of the enzyme. We solve high-resolution (2.6 Å) cryo-electron microscopy (cryo-EM) structures of the supercomplex with bound LPZS that together with microsecond molecular dynamics simulations, directed mutagenesis, and functional assays reveal key interactions that stabilize the inhibitor, but also how mutations can lead to the emergence of drug resistance. Our combined findings reveal an inhibitory mechanism of LPZS and provide a structural basis for drug development against tuberculosis.

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.

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.

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.

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.

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 (TM3^ND6) 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. 

Photosystem II (PSII), the water/plastoquinone photo-oxidoreductase, plays a key energy input role in the biosphere. , 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 at physiological O₂ concentrations with a t_1/2 of 10 s. Superoxide is formed stoichiometrically, and the reaction kinetics are controlled by the accessibility of O₂ to a binding site near , with an apparent dissociation constant of 70 ± 20 µM. Unexpectedly, 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 . These results, together with molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics calculations, indicate that electron transfer from 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 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 . This could be beneficial by oxidizing and by producing superoxide, a chemical signal for the overreduced state of the electron transfer chain.

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 Tyr_z (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.