Proton-coupled electron transfer dynamics in the alternative oxidase.

The alternative oxidase (AOX) is a membrane-bound di-iron enzyme that catalyzes O-driven quinol oxidation in the respiratory chains of plants, fungi, and several pathogenic protists of biomedical and industrial interest. Yet, despite significant biochemical and structural efforts over the last decades, the catalytic principles of AOX remain poorly understood. We develop here multi-scale quantum and classical molecular simulations in combination with biochemical experiments to address the proton-coupled electron transfer (PCET) reactions responsible for catalysis in AOX from , the causative agent of sleeping sickness.

Dissected antiporter modules establish minimal proton-conduction elements of the respiratory complex I.

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.

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 .

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 CO, 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 (H).

Electric fields control water-gated proton transfer in cytochrome oxidase.

Aerobic life is powered by membrane-bound enzymes that catalyze the transfer of electrons to oxygen and protons across a biological membrane. Cytochrome oxidase (CO) functions as a terminal electron acceptor in mitochondrial and bacterial respiratory chains, driving cellular respiration and transducing the free energy from O reduction into proton pumping. Here we show that CO creates orientated electric fields around a nonpolar cavity next to the active site, establishing a molecular switch that directs the protons along distinct pathways.

Redox-controlled reorganization and flavin strain within the ribonucleotide reductase R2b-NrdI complex monitored by serial femtosecond crystallography.

Redox reactions are central to biochemistry and are both controlled by and induce protein structural changes. Here, we describe structural rearrangements and crosstalk within the ribonucleotide reductase R2b-NrdI complex, a di-metal carboxylate-flavoprotein system, as part of the mechanism generating the essential catalytic free radical of the enzyme. Femtosecond crystallography at an X-ray free electron laser was utilized to obtain structures at room temperature in defined redox states without suffering photoreduction.

Structural basis of mammalian complex IV inhibition by steroids.

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 to oxygen, contributing to the proton motive force in the process.

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.

Functional Water Wires Catalyze Long-Range Proton Pumping in 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 (∼300 Å) 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 Å wide membrane domain.

Water-Gated Proton Transfer Dynamics in Respiratory Complex I.

The respiratory complex I transduces redox energy into an electrochemical proton gradient in aerobic respiratory chains, powering energy-requiring processes in the cell. However, despite recently resolved molecular structures, the mechanism of this gigantic enzyme remains poorly understood. By combining large-scale quantum and classical simulations with site-directed mutagenesis and biophysical experiments, we show here how the conformational state of buried ion-pairs and water molecules control the protonation dynamics in the membrane domain of complex I and establish evolutionary conserved long-range coupling elements.

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 (CO) into organic molecules. Photorespiration, however, significantly reduces the photosynthetic yields. To survive under low CO concentrations, cyanobacteria evolved unique carbon-concentration mechanisms that enhance the efficiency of photosynthetic CO fixation, for which the molecular principles have remained unknown.

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.

Molecular mechanism of polyketide shortening in anthraquinone biosynthesis of .

Anthraquinones, a widely distributed class of aromatic natural products, are produced by a type II polyketide synthase system in the Gram-negative bacterium . Heterologous expression of the anthraquinone biosynthetic gene cluster in identified AntI as an unusual lyase, catalysing terminal polyketide shortening prior to formation of the third aromatic ring. Functional and analysis of AntI using X-ray crystallography, structure-based mutagenesis, and molecular simulations revealed that AntI converts a defined octaketide to the tricyclic anthraquinone ring retro-Claisen and Dieckmann reactions.

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.

Energetics and Dynamics of Proton-Coupled Electron Transfer in the NADH/FMN Site of Respiratory Complex I.

Complex I functions as an initial electron acceptor in aerobic respiratory chains that reduces quinone and pumps protons across a biological membrane. This remarkable charge transfer process extends ca. 300 Å and it is initiated by a poorly understood proton-coupled electron transfer (PCET) reaction between nicotinamide adenine dinucleotide (NADH) and a protein-bound flavin (FMN) cofactor.

Molecular dynamics and structural models of the cyanobacterial NDH-1 complex.

NDH-1 is a gigantic redox-driven proton pump linked with respiration and cyclic electron flow in cyanobacterial cells. Based on experimentally resolved X-ray and cryo-EM structures of the respiratory complex I, we derive here molecular models of two isoforms of the cyanobacterial NDH-1 complex involved in redox-driven proton pumping (NDH-1L) and CO-fixation (NDH-1MS). Our models show distinct structural and dynamic similarities to the core architecture of the bacterial and mammalian respiratory complex I.

How inter-subunit contacts in the membrane domain of complex I affect proton transfer energetics.

The respiratory complex I is a redox-driven proton pump that employs the free energy released from quinone reduction to pump protons across its complete ca. 200 Å wide membrane domain. Despite recently resolved structures and molecular simulations, the exact mechanism for the proton transport process remains unclear.

Inhibition of Mammalian 15-Lipoxygenase by Three Ebselen-like Drugs. A QM/MM and MM/PBSA Comparative Study.

Ebselen is a potent competitive inhibitor of the active form of rabbit 15-lipoxygenase, an enzyme involved in many inflammatory diseases. Light-induced Z-to-E isomerization of the ebselen-like 2-(3-benzylidene)-3-oxo-2,3-dihydrobenzo[b]thiophene-7-carboxylic acid methyl ester (BODTCM) molecule was used to convert the weak (Z)-BOTDCM inhibitor into the (E)-isomer with much higher inhibitory capacity. In this study, the binding modes of ebselen, (E)-BOTDCM and (Z)-BOTDCM, have been analyzed to provide molecular insights on the inhibitory potency of ebselen and on the geometric-isomer specificity of (E)- and (Z)-BOTDCM inhibitors.

Mutagenesis of Sequence Determinants of Truncated Porcine ALOX15 Induces Changes in the Reaction Specificity by Altering the Catalytic Mechanism of Initial Hydrogen Abstraction.

The reaction specificity of lipoxygenases is of physiological relevance since the various oxygenation products exhibit different biological activities. Among mammalian ALOX15 orthologs there are arachidonic acid 12- and 15-lipoxygenating enzymes and recent studies suggested an evolutionary switch in that reaction specificity during late primate development. Previous reports showed that 12-lipoxygenating ALOX15 orthologs can be converted to 15-lipoxygenating enzymes by site-directed mutagenesis of some sequence determinants.

Is Regioselectivity in the Enzyme-Catalyzed Hydroperoxidation of Arachidonic Acid Necessarily Determined by Hydrogen Abstraction? The Case of Rabbit Leu597Ala/Ile663Ala ALOX15 Mutant.

Molecular dynamics simulations and quantum mechanics/molecular mechanics calculations were performed on the in silico Leu597Ala/Ile663Ala double mutant of rabbit ALOX15 (12/15 lipoxygenase). The computational results suggested that subtle steric hindrance by the conserved Leu597 and C-terminal Ile663 residues disturbed H10 abstractions in wildtype ALOX15 (which abstracts H13), but if these two bulky residues were mutated to smaller ones, H10 abstraction was no longer impeded and the regioselectivity of the initial H-abstraction step was changed. However, site-directed mutagenesis with HPLC analysis of the products of the whole oxidation process showed that the regioselectivity of the hydroperoxidation was not altered.

Computational insight into the catalytic implication of head/tail-first orientation of arachidonic acid in human 5-lipoxygenase: consequences for the positional specificity of oxygenation.

In the present work we have combined homology modeling, protein-ligand dockings, quantum mechanics/molecular mechanics calculations and molecular dynamics simulations to generate human 5-lipoxygenase (5-LOX):arachidonic acid (AA) complexes consistent with the 5-lipoxygenating activity (which implies hydrogen abstraction at the C7 position). Our results suggest that both the holo and the apo forms of human Stable 5-LOX could accommodate AA in a productive form for 5-lipoxygenation. The former, in a tail-first orientation, with the AA carboxylate end interacting with Lys409, gives the desired structures with C7 close to the Fe-OH(-) cofactor and suitable barrier heights for H7 abstraction.

Evolutionary alteration of ALOX15 specificity optimizes the biosynthesis of antiinflammatory and proresolving lipoxins.

ALOX15 (12/15-lipoxygenase) orthologs have been implicated in maturational degradation of intracellular organelles and in the biosynthesis of antiinflammatory and proresolving eicosanoids. Here we hypothesized that lower mammals (mice, rats, pigs) express 12-lipoxygenating ALOX15 orthologs. In contrast, 15-lipoxygenating isoforms are found in higher primates (orangutans, men), and these results suggest an evolution of ALOX15 specificity.

How Can Linoleic Acid Be the Preferential Substrate of the Enzyme 15-Lipoxygenase-1? A QM/MM Approach.

The most common substrate of mammalian lipoxygenases (LOXs) is arachidonic acid (AA). However, 15-LOXs can present dual substrate specificity. These LOXs catalyze the peroxidation of AA, initiated by a H-abstraction step (mainly H13-abstraction) by the Fe(III)-OH(-) cofactor, and the peroxidation of linoleic acid (LA) after H11-abstraction.