RESUMO
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.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons , Prótons , Aerobiose , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/química , Oxigênio/metabolismo , Água/químicaRESUMO
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.
Assuntos
Diosgenina , Complexo IV da Cadeia de Transporte de Elétrons , Esteroides , Animais , Sítios de Ligação , Domínio Catalítico/efeitos dos fármacos , Bovinos , Diosgenina/farmacologia , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/antagonistas & inibidores , Complexo IV da Cadeia de Transporte de Elétrons/química , Oxirredução , Oxigênio/metabolismo , Conformação Proteica , Prótons , Esteroides/química , Esteroides/farmacologia , EsteróisRESUMO
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.
Assuntos
Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Prótons , Animais , Sítios de Ligação , Transporte Biológico , Respiração Celular , Microscopia Crioeletrônica , Complexo I de Transporte de Elétrons/genética , Metabolismo Energético , Humanos , Doenças Mitocondriais/genética , Membranas Mitocondriais/química , Membranas Mitocondriais/metabolismo , Simulação de Dinâmica Molecular , Mutação , Oxirredução , Conformação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Quinonas/química , Quinonas/metabolismo , Água/química , Água/metabolismoRESUMO
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 â 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.
Assuntos
Complexo I de Transporte de Elétrons , Prótons , Complexo I de Transporte de Elétrons/metabolismo , Oxirredução , Transporte de Elétrons , Quinonas , Bombas de Próton/metabolismo , CatáliseRESUMO
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.
Assuntos
Elétrons , Hidrogenase , Hidrogenase/química , NAD/metabolismo , Microscopia Crioeletrônica , Ferredoxinas/química , Oxirredução , Hidrogênio/química , Transporte de ElétronsRESUMO
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 (â¼300 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. 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.
Assuntos
Biocatálise , Complexo I de Transporte de Elétrons/metabolismo , Simulação de Dinâmica Molecular , Água/metabolismo , Transporte de Elétrons , Complexo I de Transporte de Elétrons/química , Teoria Quântica , Eletricidade Estática , TermodinâmicaRESUMO
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. We suggest that an electrostatic wave propagates in forward and reverse directions across the 200 Å long membrane domain during enzyme turnover, without significant dissipation of energy. Our findings demonstrate molecular principles that enable efficient long-range proton-electron coupling (PCET) and how perturbation of this PCET machinery may lead to development of mitochondrial disease.
Assuntos
Complexo I de Transporte de Elétrons/metabolismo , Simulação de Dinâmica Molecular , Prótons , Água/metabolismo , Teoria da Densidade Funcional , Complexo I de Transporte de Elétrons/química , Oxirredução , Água/químicaRESUMO
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. We combine here large-scale density functional theory calculations and quantum/classical models with atomistic molecular dynamics simulations to probe the energetics and dynamics of the NADH-driven PCET reaction in complex I. We find that the reaction takes place by concerted hydrogen atom (Hâ¢) transfer that couples to an electron transfer (eT) between the aromatic ring systems of the cofactors and further triggers reduction of the nearby FeS centers. In bacterial, Escherichia coli-like complex I isoforms, reduction of the N1a FeS center increases the binding affinity of the oxidized NAD+ that prevents the nucleotide from leaving prematurely. This electrostatic trapping could provide a protective gating mechanism against reactive oxygen species formation. We also find that proton transfer from the transient FMNH⢠to a nearby conserved glutamate (Glu97) residue favors eT from N1a onward along the FeS chain and modulates the binding of a new NADH molecule. The PCET in complex I isoforms with low-potential N1a centers is also discussed. On the basis of our combined results, we propose a putative mechanistic model for the NADH-driven proton/electron-transfer reaction in complex I.
Assuntos
Complexo I de Transporte de Elétrons/química , Complexo I de Transporte de Elétrons/metabolismo , Mononucleotídeo de Flavina/metabolismo , NAD/metabolismo , Prótons , Teoria da Densidade Funcional , Transporte de Elétrons , Isoenzimas/química , Isoenzimas/metabolismo , Simulação de Dinâmica Molecular , Conformação Proteica , TermodinâmicaRESUMO
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. To test this hypothesis we first cloned and characterized ALOX15 orthologs of selected Catarrhini representing different stages of late primate evolution and found that higher primates (men, chimpanzees) express 15-lipoxygenating orthologs. In contrast, lower primates (baboons, rhesus monkeys) express 12-lipoxygenating enzymes. Gibbons, which are flanked in evolution by rhesus monkeys (12-lipoxygenating ALOX15) and orangutans (15-lipoxygenating ALOX15), express an ALOX15 ortholog with pronounced dual specificity. To explore the driving force for this evolutionary alterations, we quantified the lipoxin synthase activity of 12-lipoxygenating (rhesus monkey, mouse, rat, pig, humIle418Ala) and 15-lipoxygenating (man, chimpanzee, orangutan, rabbit, ratLeu353Phe) ALOX15 variants and found that, when normalized to their arachidonic acid oxygenase activities, the lipoxin synthase activities of 15-lipoxygenating ALOX15 variants were more than fivefold higher (P < 0.01) [corrected]. Comparative molecular dynamics simulations and quantum mechanics/molecular mechanics calculations indicated that, for the 15-lipoxygenating rabbit ALOX15, the energy barrier for C13-hydrogen abstraction (15-lipoxygenation) was 17 kJ/mol lower than for arachidonic acid 12-lipoxygenation. In contrast, for the 12-lipoxygenating Ile418Ala mutant, the energy barrier for 15-lipoxygenation was 10 kJ/mol higher than for 12-lipoxygenation. Taken together, our data suggest an evolution of ALOX15 specificity, which is aimed at optimizing the biosynthetic capacity for antiinflammatory and proresolving lipoxins.
Assuntos
Araquidonato 15-Lipoxigenase/genética , Araquidonato 15-Lipoxigenase/metabolismo , Evolução Molecular , Lipoxinas/biossíntese , Animais , Anti-Inflamatórios/química , Anti-Inflamatórios/metabolismo , Araquidonato 15-Lipoxigenase/química , Domínio Catalítico , Humanos , Lipoxinas/química , Camundongos , Mutação , Primatas , Coelhos , Ratos , Especificidade da Espécie , Especificidade por Substrato , SuínosRESUMO
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. 200â¯Å wide membrane domain. Despite recently resolved structures and molecular simulations, the exact mechanism for the proton transport process remains unclear. Here we combine large-scale molecular simulations with quantum chemical density functional theory (DFT) models to study how contacts between neighboring antiporter-like subunits in the membrane domain of complex I affect the proton transfer energetics. Our combined results suggest that opening of conserved Lys/Glu ion pairs within each antiporter-like subunit modulates the barrier for the lateral proton transfer reactions. Our work provides a mechanistic suggestion for key coupling effects in the long-range force propagation process of complex I.
Assuntos
Membrana Celular/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Metabolismo Energético , Prótons , Thermus thermophilus/metabolismo , Transporte de Elétrons , Modelos Moleculares , Simulação de Dinâmica Molecular , Oxirredução , Conformação Proteica , Domínios Proteicos , Subunidades Proteicas , Bombas de PrótonRESUMO
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. Unfortunately, the molecular basis for those alterations are not well understood. Here, the arachidonic acid 12-lipoxygenating N-terminal truncation variant of pig ALOX15, for which a crystal structure is available, was used to explore the catalytic mechanism of the specificity switch induced by mutagenesis of Val418 and Val419 sequence determinants. We found that Val418Ile+Val419Met double mutant is dominantly 15-lipoxygenating. Docking and MD simulations, and quantum mechanics/molecular mechanics calculations indicated that the wildtype energy barrier for arachidonic acid 15-lipoxygenation is 3.4â kcal mol-1 higher than for 12-lipoxygenation. In contrast, for the Val418Ile+Val419Met double mutant the energy barrier for 12-lipoxygenation is 6.0â kcal mol-1 higher than for 15-lipoxygenation. Our data suggest that enzyme-substrate complex geometries determine the value of these energy barriers and, as a consequence, the reaction specificity of ALOX15 orthologs.
RESUMO
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. The inhibitor-enzyme structures obtained from docking and molecular dynamics simulations as well as from QM/MM calculations show that the inhibitor molecules are not coordinated to the nonheme iron in the active site. Thermal motion allows ebselen and (E)-BOTDCM to visit a wide range of the configurational space competing with the polyunsaturated fatty acid for binding at the active site. Both molecules present similar MM/PBSA binding free energies. The energy penalty for the bigger geometric deformation undergone by (E)-BODTCM would explain its lower inhibitor potency. The (Z)-isomer is the weakest inhibitor because thermal motion moves it to a region very far from the first coordination sphere of Fe, where it could not compete with the fatty acid substrate.
Assuntos
Araquidonato 15-Lipoxigenase/metabolismo , Azóis/farmacologia , Compostos Férricos/farmacologia , Inibidores de Lipoxigenase/farmacologia , Compostos Organosselênicos/farmacologia , Teoria Quântica , Termodinâmica , Animais , Azóis/síntese química , Azóis/química , Compostos Férricos/síntese química , Compostos Férricos/química , Isoindóis , Ligantes , Inibidores de Lipoxigenase/síntese química , Inibidores de Lipoxigenase/química , Modelos Moleculares , Estrutura Molecular , Compostos Organosselênicos/síntese química , Compostos Organosselênicos/química , CoelhosRESUMO
Molecular dynamics simulations and quantum mechanics/molecular mechanics calculations were performed on the inâ 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. This disagreement may be explained by the conformational reorganization of the system needed to rotate the -OO. group from an antarafacial to a suprafacial arrangement prior to back-hydrogen transfer. After H10 abstraction and O2 insertion, the evolution of the peroxy radical at C12 was sterically impeded, whereas peroxyl group rotation at C15 (after H13 abstraction) could easily evolve to a suprafacial arrangement, which thus led to the final product. For this reason, the global regiospecificity was not affected in the mutant. These findings exemplify that the regioselectivity of initial hydrogen abstraction and the regioselectivity of the final product do not necessarily coincide (in fact, they can be opposite) for the hydroperoxidation of arachidonic acid catalyzed by a lipoxygenase.
Assuntos
Araquidonato 12-Lipoxigenase/genética , Araquidonato 12-Lipoxigenase/metabolismo , Araquidonato 15-Lipoxigenase/genética , Araquidonato 15-Lipoxigenase/metabolismo , Ácido Araquidônico/metabolismo , Biocatálise , Hidrogênio/metabolismo , Animais , Ácido Araquidônico/química , Hidrogênio/química , Simulação de Dinâmica Molecular , Mutação , Oxirredução , Teoria Quântica , Coelhos , EstereoisomerismoRESUMO
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. Only when using the apo form structure, a head-first orientation with the AA carboxylate close to His600 (a residue recently proposed as essential for AA positioning) is obtained in the docking calculations. However, the calculated barrier heights for this head-first orientation are in principle consistent with 5-LOX specificity, but also with 12/8 regioselectivity. Finally, long MD simulations give support to the recent hypothesis that the Phe177 + Tyr181 pair needs to close the active site access during the chemical reaction, and suggest that in the case of a head-first orientation Phe177 may be the residue interacting with the AA carboxylate.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Ácido Araquidônico/metabolismo , Sítios de Ligação , Simulação de Dinâmica Molecular , Catálise , Humanos , HidrogênioRESUMO
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 Bacillus cereus 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. Together with density functional theory calculations, we show that the flavin is under steric strain in the R2b-NrdI protein complex, likely tuning its redox properties to promote superoxide generation. Moreover, a binding site in close vicinity to the expected flavin O2 interaction site is observed to be controlled by the redox state of the flavin and linked to the channel proposed to funnel the produced superoxide species from NrdI to the di-manganese site in protein R2b. These specific features are coupled to further structural changes around the R2b-NrdI interaction surface. The mechanistic implications for the control of reactive oxygen species and radical generation in protein R2b are discussed.
Assuntos
Ribonucleotídeo Redutases , Cristalografia por Raios X , Flavinas/metabolismo , Oxirredução , Ribonucleotídeo Redutases/química , SuperóxidosRESUMO
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.
Assuntos
Carbono/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Fotossíntese , Bombas de Próton/metabolismo , Dióxido de Carbono/metabolismo , Domínio Catalítico , Complexo I de Transporte de Elétrons/química , Oxirredução , Eletricidade Estática , Thermus/metabolismo , Água/metabolismoRESUMO
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 CO2-fixation (NDH-1MS). Our models show distinct structural and dynamic similarities to the core architecture of the bacterial and mammalian respiratory complex I. We identify putative plastoquinone-binding sites that are coupled by an electrostatic wire to the proton pumping elements in the membrane domain of the enzyme. Molecular simulations suggest that the NDH-1L isoform undergoes large-scale hydration changes that support proton-pumping within antiporter-like subunits, whereas the terminal subunit of the NDH-1MS isoform lacks such structural motifs. Our work provides a putative molecular blueprint for the complex I-analogue in the photosynthetic energy transduction machinery and demonstrates that general mechanistic features of the long-range proton-pumping machinery are evolutionary conserved in the complex I-superfamily.
Assuntos
Cianobactérias/enzimologia , Simulação de Dinâmica Molecular , NADH Desidrogenase/química , Antiporters , Proteínas de Bactérias/química , Sítios de Ligação , Complexo I de Transporte de Elétrons/química , Transferência de Energia , Estrutura Molecular , Fotossíntese , Plastoquinona/metabolismo , Isoformas de Proteínas , Subunidades Proteicas , Bombas de PrótonRESUMO
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.
Assuntos
Benzoquinonas/química , Complexo I de Transporte de Elétrons/química , Modelos Químicos , Catálise , Teoria da Densidade Funcional , Modelos Moleculares , Simulação de Dinâmica Molecular , Teoria Quântica , Eletricidade EstáticaRESUMO
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.
Assuntos
Eletricidade , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Animais , Bovinos , Complexo IV da Cadeia de Transporte de Elétrons/química , Oxirredução , Fotossíntese , Prótons , Termodinâmica , Água/químicaRESUMO
Anthraquinones, a widely distributed class of aromatic natural products, are produced by a type II polyketide synthase system in the Gram-negative bacterium Photorhabdus luminescens. Heterologous expression of the antABCDEFGHI anthraquinone biosynthetic gene cluster in Escherichia coli identified AntI as an unusual lyase, catalysing terminal polyketide shortening prior to formation of the third aromatic ring. Functional in vitro and in vivo 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 via retro-Claisen and Dieckmann reactions. Thus, AntI catalyses a so far unobserved multistep reaction in this PKS system.