RESUMEN
The interconversion between fumarate and succinate is fundamental to the energy metabolism of nearly all organisms. This redox reaction is catalyzed by a large family of enzymes, fumarate reductases and succinate dehydrogenases, using hydride and proton transfers from a flavin cofactor and a conserved Arg side-chain. These flavoenzymes also have substantial biomedical and biotechnological importance. Therefore, a detailed understanding of their catalytic mechanisms is valuable. Here, calibrated electronic structure calculations in a cluster model of the active site of the Fcc3 fumarate reductase were employed to investigate various reaction pathways and possible intermediates in the enzymatic environment and to dissect interactions that contribute to catalysis of fumarate reduction. Carbanion, covalent adduct, carbocation, and radical intermediates were examined. Significantly lower barriers were obtained for mechanisms via carbanion intermediates, with similar activation energies for hydride and proton transfers. Interestingly, the carbanion bound to the active site is best described as an enolate. Hydride transfer is stabilized by a preorganized charge dipole in the active site and by the restriction of the C1-C2 bond in a twisted conformation of the otherwise planar fumarate dianion. But, protonation of a fumarate carboxylate and quantum tunneling effects are not critical for catalysis of the hydride transfer. Calculations also suggest that the driving force for enzyme turnover is provided by regeneration of the catalytic Arg, either coupled with flavin reduction and decomposition of a proposed transient state or directly from the solvent. The detailed mechanistic description of enzymatic reduction of fumarate provided here clarifies previous contradictory views and provides new insights into catalysis by essential flavoenzyme reductases and dehydrogenases.
Asunto(s)
Protones , Succinatos , Oxidación-Reducción , Catálisis , Fumaratos/metabolismo , Flavinas/metabolismo , CinéticaRESUMEN
Proteins equipped with flavin adenine dinucleotides (FAD) or flavin mononucleotides (FMN) are named flavoproteins and constitute about 1% of all existing proteins. They catalyze redox, acid-base and photochemical reactions in a variety of biochemical phenomena that goes from energy metabolism to DNA repair and light sensing. The versatility observed in flavoproteins is ultimately a balance of flavin intrinsic properties modulated by a protein environment. This thesis aims to investigate how flavoproteins work by systematic evaluating flavin properties and reactivity. In particular, the mechanism of fumarate reduction by the flavoenzyme fumarate reductase Fcc3 was determined. Electronic-structure calculations were used for this task based on rigorous calibration with experimental data and error assessment. Flavin properties at chemical accuracy were obtained with single reference coupled-cluster CCSD(T) calculations at the complete basis set limit. Density functional theory was demonstrated an excellent alternative with lower computational costs and slightly less accuracy. Flavin protonation and tautomerism were shown to be important modulators of flavin properties and reactivity, with the possibility of various tautomers existing at neutral pH. Regarding flavin redox properties, an analysis based on multiconfigurational wave function weights was proposed for categorizing flavin redox reactions as hydride or hydrogen-atom transfers. This analysis is an upgrade over traditional partial charges methods and can be applied not only to flavin reactions but to any protoncoupled electron transfer. In the investigation of the enzymatic mechanism of fumarate reduction, the reaction was determined as a nucleophilic addition by hydride transfer with carbanion formation. Fumarate reductase employs electrostatic catalysis in contrast to previous proposals of substrate straining and general-acid catalysis. Also, hydride transfer was shown to be vibronically adiabatic with low tunneling contribution. These findings give new insights into the mechanisms of fumarate reductases and provide a framework for future computational studies of flavoproteins in general. The analyses and benchmark studies presented can be used to build better models of properties and reactivity of flavins and flavoproteins
Proteínas equipadas com dinucleotídeos de flavina-adenina (FAD) e mononucleotídeos de flavina (FMN) são chamadas flavoproteínas e constituem cerca de 1% de todas as proteínas existentes. Elas catalisam reações redox, ácido-base e fotoquímicas numa variedade de fenômenos bioquímicos que vão desde o metabolismo energético até reparo de DNA e captação de luz. A versatilidade observada em flavoproteínas é em última instância um balanço das propriedades intrínsecas de flavinas moduladas por um ambiente proteico. Esta tese busca investigar como flavoproteínas funcionam através de avaliações sistemáticas de propriedades e reatividade de flavinas. Em particular, o mecanismo de redução de fumarato pela flavoenzima fumarato redutase Fcc3 foi determinado. Cálculos de estrutura eletrônica foram usados para esta tarefa com base em rigorosa calibração com dados experimentais e avaliação de erros. As propriedades de flavinas foram determinadas com acurácia química com cálculos monoconfiguracionais de coupled-cluster CCSD(T) no limite de conjunto base completo. A teoria do funcional da densidade mostrou-se uma alternativa excelente com menor custo computacional e um pouco menos de acurácia. Protonação e tautomerismo de flavinas mostraram-se moduladores importantes de suas propriedades e reatividade, com a possibilidade de vários tautômeros existirem em pH neutro. Em relação às propriedades redox de flavinas, uma análise baseada nos pesos de funções de onda multiconfiguracionais foi proposta para categorizar as reações redox de flavinas como transferências de hidreto ou hidrogênio. Esta análise é uma melhoria em relação aos métodos tradicionais de cargas parciais e pode ser aplicada não apenas para reações de flavinas mas para qualquer transferência de próton acoplada a elétrons. Na investigação do mecanismo enzimático de redução de fumarato, a reação foi designada como uma adição nucleofílica por transferência de hidreto e formação de carbânion. A fumarato redutase usa catálise eletrostática diferentemente de prospostas anteriores envolvendo distorção do substrato e catálise ácida geral. Além disso, a transferência de hidreto mostrou-se vibronicamente adiabática com pouca contribuição de tunelamento. Estas descobertas abrem novas perspectivas sobre os mecanismos de fumarato redutases e fornecem uma base para estudos computacionais futuros sobre flavoproteínas em geral. As análises e estudos comparativos apresentados podem ser usados para construir melhores modelos para propriedades e reatividade de flavinas e flavoproteínas
Asunto(s)
Estudio Comparativo , Flavinas/análisis , Flavoproteínas/análisis , Cálculos/química , Electricidad Estática/efectos adversos , FumaratosRESUMEN
Flavins are employed as redox cofactors and chromophores in a plethora of flavoenzymes. Their versatility is an outcome of intrinsic molecular properties of the isoalloxazine ring modulated by the protein scaffold and surrounding solvent. Thus, an investigation of isolated flavins with high-level electronic-structure methods and with error assessment of the calculated properties will contribute to building better models of flavin reactivity. Here, we benchmarked ground-state properties such as electron affinity, gas-phase basicity, dipole moment, torsion energy, and tautomer stability for lumiflavins in all biologically relevant oxidation and charge states. Overall, multiconfigurational effects are small and chemical accuracy is achieved by coupled-cluster treatments of energetic properties. Augmented basis sets and extrapolations to the complete basis-set limit are necessary for consistent agreement with experimental energetics. Among DFT functionals tested, M06-2X shows the best performance for most properties, except gas-phase basicity, in which M06 and CAM-B3LYP perform better. Moreover, dipole moments of radical flavins show large deviations for all functionals studied. Tautomers with noncanonical protonation states are significantly populated at normal temperatures, adding to the complexity of modeling flavins. These results will guide future computational studies of flavoproteins and flavin chemistry by indicating the limitations of electronic-structure methodologies and the contributions of multiple tautomeric states.
Asunto(s)
Flavinas , Flavinas/química , Oxidación-ReducciónRESUMEN
Human metallothionein (MT) is a small-size yet efficient metal-binding protein, playing an essential role in metal homeostasis and heavy metal detoxification. MT contains two domains, each forming a polynuclear metal cluster with an exquisite hexatomic ring structure. The apoprotein is intrinsically disordered, which may strongly influence the clusters and the metal-thiolate (M-S) bonds, leading to a highly dynamic structure. However, these features are challenging to identify due to the transient nature of these species. The individual signal from dynamic conformations with different states of the cluster and M-S bond will be averaged and blurred in classic ensemble measurement. To circumvent these problems, we combined a single-molecule approach and multiscale molecular simulations to investigate the rupture mechanism and chemical stability of the metal cluster by a single MT molecule, focusing on the Zn4S11 cluster in the α domain upon unfolding. Unusual multiple unfolding pathways and intermediates are observed for both domains, corresponding to different combinations of M-S bond rupture. None of the pathways is clearly preferred suggesting that unfolding proceeds from the distribution of protein conformational substates with similar M-S bond strengths. Simulations indicate that the metal cluster may rearrange, forming and breaking metal-thiolate bonds even when MT is folded independently of large protein backbone reconfiguration. Thus, a highly dynamic polynuclear metal cluster with multiple conformational states is revealed in MT, responsible for the binding promiscuity and diverse cellular functions of this metal-carrier protein.
RESUMEN
Cytochrome bc1 is a fundamental enzyme for cellular respiration and photosynthesis. This dimeric protein complex catalyzes a proton-coupled electron transfer (PCET) from the reduced coenzyme-Q substrate (Q) to a bimetallic iron-sulfur cluster in the Qo active site. Herein, we combine molecular dynamics simulations of the complete cytochrome bc1 protein with electronic-structure calculations of truncated models and a semiclassical tunneling theory to investigate the electron-proton adiabaticity of the initial reaction catalyzed in the Qo site. After sampling possible orientations between the Q substrate and a histidine side chain that functions as hydrogen acceptor, we find that a truncated model composed by ubiquinol-methyl and imidazole-iron(III)-sulfide captures the expected changes in oxidation and spin states of the electron donor and acceptor. Diabatic electronic surfaces obtained for this model with multiconfigurational wave function calculations demonstrate that this reaction is electronic nonadiabatic, and proton tunneling is faster than mixing of electronic configurations. These results indicate the formalism that should be used to calculate vibronic couplings and kinetic parameters for the initial reaction in the Qo site of cytochrome bc1. This framework for molecular simulation may also be applied to investigate other PCET reactions in the Q-cycle or in various metalloproteins that catalyze proton translocation coupled to redox processes.
Asunto(s)
Electrones , Protones , Respiración de la Célula , Citocromos , Transporte de Electrón , Compuestos Férricos , Oxidación-ReducciónRESUMEN
Flavins are versatile biological cofactors which catalyze proton-coupled electron transfers (PCET) with varying number and coupling of electrons. Flavin-mediated oxidations of nicotinamide adenine dinucleotide (NADH) and of succinate, initial redox reactions in cellular respiration, were examined here with multiconfigurational quantum chemical calculations and a simple analysis of the wave function proposed to quantify electron transfer along the proton reaction coordinate. The mechanism of NADH oxidation is a prototypical hydride transfer, with two electrons moving concerted with the proton to the same acceptor group. However, succinate oxidation depends on the elimination step and can proceed through the transfer of a hydride or hydrogen atom, with proton and electrons moving to different groups in both cases. These results help to determine the mechanism of fundamental but still debated biochemical reactions and illustrate a new diagnostic tool for electron transfer that can be useful to characterize a broad class of PCET processes.
Asunto(s)
Flavinas , Hidrógeno , Dinitrocresoles , Transporte de Electrón , Oxidación-ReducciónRESUMEN
Iron-sulfur (FeS) clusters are essential metal cofactors involved in a wide variety of biological functions. Their catalytic efficiency, biosynthesis, and regulation depend on FeS stability in aqueous solution. Here, molecular modeling is used to investigate the hydrolysis of an oxidized (ferric) mononuclear FeS cluster by bare dissociation and water substitution mechanisms in neutral and acidic solution. First, approximate electronic structure descriptions of FeS reactions by density functional theory are validated against high-level wave function CCSD(T) calculations. Solvation contributions are included by an all-atom model with hybrid quantum chemical/molecular mechanical (QM/MM) potentials and enhanced sampling molecular dynamics simulations. The free energy profile obtained for FeS cluster hydrolysis indicates that the hybrid functional M06 together with an implicit solvent correction capture the most important aspects of FeS cluster reactivity in aqueous solution. Then, 20 reaction channels leading to two consecutive Fe-S bond ruptures were explored with this calibrated model. For all protonation states, nucleophilic substitution with concerted bond breaking and forming to iron is the preferred mechanism, both kinetic and thermodynamically. In neutral solution, proton transfer from water to the sulfur leaving group is also concerted. Dissociative reactions show higher barriers and will not be relevant for FeS reactivity when exposed to solvent. These hydrolysis mechanisms may help to explain the stability and catalytic mechanisms of FeS clusters of multiple sizes and proteins.