Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotide (RNA) to deoxyribonucleotide (DNA) building blocks initiated by a long-range (>30 Å) proton-coupled electron transfer (PCET) by mechanistic principles that remain much debated. By combining multiscale quantum and classical simulations with directed mutagenesis, X-ray crystallography, and vibrational and electron paramagnetic resonance spectroscopy, we elucidate here the molecular principles underlying how metal-free RNRs initiate the long-range PCET process by creating a highly stable 3,4-dihydroxyphenylalanine (DOPA) initiator radical. We show that DOPA• is redox-tuned by a low-barrier hydrogen bond (LBHB), with a delocalized proton that provides the catalytic power for the ribonucleotide reduction. We find that the LBHB couples to an extended hydrogen-bonded network, with distant mutations resulting in the loss of radical formation, and providing key molecular insight into the long-range radical transport mechanism in RNRs. On a general level, our findings support the direct involvement of LBHB in protein chemistry and the importance of quantum effects in enzyme catalysis.

Mitochondrial membranes harbor the electron transport chain (ETC) that powers oxidative phosphorylation (OXPHOS) and drives the synthesis of ATP. Yet, under physiological conditions, the OXPHOS proteins operate as higher-order supercomplex (SC) assemblies, although their functional role remains poorly understood and much debated. By combining large-scale atomistic and coarse-grained molecular simulations with analysis of cryo-electron microscopic data and statistical as well as kinetic models, we show here that the formation of the mammalian I/III₂ supercomplex reduces the molecular strain of inner mitochondrial membranes by altering the local membrane thickness and leading to an accumulation of both cardiolipin and quinone around specific regions of the SC. We find that the SC assembly also affects the global motion of the individual ETC proteins with possible functional consequences. On a general level, our findings suggest that molecular crowding and strain effects provide a thermodynamic driving force for the SC formation, with a possible flux enhancement in crowded biological membranes under constrained respiratory conditions.

The bioenergetic complexes of energy-transducing membranes generate a proton current that powers the synthesis of adenosine triphosphate. Yet, since the early days of the chemiosmotic theory, it has remained elusive and much debated whether the proton motive force (PMF) delocalizes into the bulk solvent surrounding the energy-transducing membrane or if the thermodynamic force is exerted as a localized proton current along the membrane surface. To elucidate the molecular principles underlying protonation dynamics at biological membranes, we combine here proteoliposome experiments with fluorescence correlation spectroscopy and multiscale molecular simulations. We show that ubiquinone (Q10), which is an essential electron carrier of inner mitochondrial membranes, interacts with protons at the membrane, and alters the rate of the protonation reactions along the surface. We find that physiological Q10 concentrations increase the integrity of the liposome membranes to sustain a PMF and enhance the rate of surface protonation reactions of lipid-conjugated pH-sensitive fluorophores, occurring on a microsecond timescale. Our multiscale simulations reveal that the quinone headgroup localizes at the membrane surface and stabilizes protonated water species by cation–π and hydrogen-bonded interactions amplifying the proton exchange on the surface relative to the bulk solvent. We suggest that in addition to the well-established role of quinones as redox mediators in energy-transducing membranes, Q10 also promotes the proton-collecting antenna effect, mediating proton exchange along the membrane and supporting a local proton circuit model. Our combined findings provide molecular insight into propagation of proton currents along biological membranes and reveal key principles underlying the energy conversion mechanisms in biology.

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.

Photosystem II (PSII) catalyzes light-driven water oxidation that releases dioxygen into our atmosphere and provides the electrons needed for the synthesis of biomass. The catalysis occurs in the oxygen-evolving oxo-manganese-calcium (Mn₄O₅Ca) cluster that drives the oxidation and deprotonation of substrate water molecules leading to the O₂ formation. However, despite recent advances, the mechanism of these reactions remains unclear and much debated. Here, we show that the light-driven Tyr161_D1 (Y_z) oxidation adjacent to the Mn₄O₅Ca cluster, decreases the barrier for proton transfer from the putative substrate water molecule (W3/W_x) to Glu310_D2, accessible to the luminal bulk. By combining hybrid quantum/classical (QM/MM) free energy calculations with atomistic molecular dynamics simulations, we probe the energetics of the proton transfer along the Cl1 pathway. We demonstrate that the proton transfer occurs via water molecules and a cluster of conserved carboxylates, driven by redox-triggered electric fields directed along the pathway. Glu65_D1 establishes a local molecular gate that controls the proton transfer to the luminal bulk, while Glu312_D2 acts as a local proton storage site. The identified gating region could be important in preventing backflow of protons to the Mn₄O₅Ca cluster. The structural changes, derived here based on the dark-state PSII structure, strongly support recent time-resolved X-ray free electron laser data of the S₃ → S₄ transition (Bhowmick et al. Nature 617, 2023) and reveal the mechanistic basis underlying deprotonation of the substrate water molecules. Our findings provide insight into the water oxidation mechanism of PSII and show how the interplay between redox-triggered electric fields, ion-pairs, and hydration effects control proton transport reactions.

Water-mediated proton transfer reactions are central for catalytic processes in a wide range of biochemical systems, ranging from biological energy conversion to chemical transformations in the metabolism. Yet, the accurate computational treatment of such complex biochemical reactions is highly challenging and requires the application of multiscale methods, in particular hybrid quantum/classical (QM/MM) approaches combined with free energy simulations. Here, we combine the unique exploration power of new advanced sampling methods with density functional theory (DFT)-based QM/MM free energy methods for multiscale simulations of long-range protonation dynamics in biological systems. In this regard, we show that combining multiple walkers/well-tempered metadynamics with an extended system adaptive biasing force method (MWE) provides a powerful approach for exploration of water-mediated proton transfer reactions in complex biochemical systems. We compare and combine the MWE method also with QM/MM umbrella sampling and explore the sampling of the free energy landscape with both geometric (linear combination of proton transfer distances) and physical (center of excess charge) reaction coordinates and show how these affect the convergence of the potential of mean force (PMF) and the activation free energy. We find that the QM/MM-MWE method can efficiently explore both direct and water-mediated proton transfer pathways together with forward and reverse hole transfer mechanisms in the highly complex proton channel of respiratory Complex I, while the QM/MM-US approach shows a systematic convergence of selected long-range proton transfer pathways. In this regard, we show that the PMF along multiple proton transfer pathways is recovered by combining the strengths of both approaches in a QM/MM-MWE/focused US (FUS) scheme and reveals new mechanistic insight into the proton transfer principles of Complex I. Our findings provide a promising basis for the quantitative multiscale simulations of long-range proton transfer reactions in biological systems. 

Cellular function is powered by mitochondria through an energy conversion process known as oxidative phosphorylation. Central to this process is respiratory complex I, an enzyme that couples NADH oxidation with ubiquinone reduction and the pumping of protons across the inner mitochondrial membrane. In this thesis, the mechanistic principles of complex I were investigated using multi-scale simulations, including atomistic molecular dynamics simulations and hybrid quantum/classical mechanics (QM/MM) calculations. We found that complex I drives quinone reduction and proton pumping through a network of buried charged residues. These residues couple protonation changes to conformational shifts, electrostatic interactions, and modulations of the hydration dynamics. Additionally, we expanded the applicability of QM/MM to long-range protonation dynamics by developing a novel sampling scheme. This scheme combines advanced sampling methods with a general reaction coordinate to provide a quantitative description of hydration dynamics and conformational changes during proton transfer reactions, which are indispensable for understanding the function of the respiratory enzymes. We further investigated the molecular details of how and why respiratory complexes cluster together to form supercomplexes. Our findings indicate that membrane proteins alter the membrane properties and introduce strain, which could drive the formation of these assemblies. The combined mechanistic findings of this thesis enhance our understanding of respiratory complex I and supercomplexes and their underlying proton transfer reactions, conformational changes, and enzymatic activity.

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.

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. 

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.