RESUMO
The conversion of solar energy into hydrogen represents a highly attractive strategy for the production of renewable energies. Photosynthetic microorganisms have the ability to produce H2 from sunlight but several obstacles must be overcome before obtaining a sustainable and efficient H2 production system. Cyanobacteria harbor [NiFe] hydrogenases required for the consumption of H2. As a result, their H2 production rates are low, which makes them not suitable for a high yield production. On the other hand, [FeFe] enzymes originating from anaerobic organisms such as Clostridium exhibit much higher H2 production activities, but their sensitivity to O2 inhibition impairs their use in photosynthetic organisms. To reach such a goal, it is therefore important to protect the hydrogenase from O2. The diazotrophic filamentous cyanobacteria protect their nitrogenases from O2 by differentiating micro-oxic cells called heterocysts. Producing [FeFe] hydrogenase in the heterocyst is an attractive strategy to take advantage of their potential in a photosynthetic microorganism. Here, we present a biological engineering approach for producing an active [FeFe] hydrogenase (HydA) from Clostridium acetobutylicum in the heterocysts of the filamentous cyanobacterium Nostoc PCC7120. To further decrease the O2 amount inside the heterocyst, the GlbN cyanoglobin from Nostoc commune was coproduced with HydA in the heterocyst. The engineered strain produced 400 µmol-H2 per mg Chlorophyll a, which represents 20-fold the amount produced by the wild type strain. This result is a clear demonstration that it is possible to associate oxygenic photosynthesis with H2 production by an O2-sensitive hydrogenase.
Assuntos
Clostridium acetobutylicum/enzimologia , Hidrogênio/metabolismo , Hidrogenase/genética , Hidrogenase/metabolismo , Microbiologia Industrial/métodos , Nostoc/genética , Organismos Geneticamente Modificados/genética , Organismos Geneticamente Modificados/metabolismoRESUMO
Shewanella species are facultative anaerobic bacteria that colonize redox-stratified habitats where O2 and nutrient concentrations fluctuate. The model species Shewanella oneidensis MR-1 possesses genes coding for three terminal oxidases that can perform O2 respiration: a bd-type quinol oxidase and cytochrome c oxidases of the cbb3-type and the A-type. Whereas the bd- and cbb3-type oxidases are routinely detected, evidence for the expression of the A-type enzyme has so far been lacking. Here, we investigated the effect of nutrient starvation on the expression of these terminal oxidases under different O2 tensions. Our results reveal that the bd-type oxidase plays a significant role under nutrient starvation in aerobic conditions. The expression of the cbb3-type oxidase is also modulated by the nutrient composition of the medium and increases especially under iron-deficiency in exponentially growing cells. Most importantly, under conditions of carbon depletion, high O2 and stationary-growth, we report for the first time the expression of the A-type oxidase in S. oneidensis, indicating that this terminal oxidase is not functionally lost. The physiological role of the A-type oxidase in energy conservation and in the adaptation of S. oneidensis to redox-stratified environments is discussed.
Assuntos
Proteínas de Bactérias/biossíntese , Complexo IV da Cadeia de Transporte de Elétrons/biossíntese , Regulação Bacteriana da Expressão Gênica/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Shewanella/enzimologia , Proteínas de Bactérias/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Consumo de Oxigênio/fisiologia , Shewanella/genéticaRESUMO
Catalytically inactive oxidized O2-sensitive [NiFe]-hydrogenases are characterized by a mixture of the paramagnetic Ni-A and Ni-B states. Upon O2 exposure, enzymes in a partially reduced state preferentially form the unready Ni-A state. Because partial O2 reduction should generate a peroxide intermediate, this species was previously assigned to the elongated Ni-Fe bridging electron density observed for preparations of [NiFe]-hydrogenases known to contain the Ni-A state. However, this proposition has been challenged based on the stability of this state to UV light exposure and the possibility of generating it anaerobically under either chemical or electrochemical oxidizing conditions. Consequently, we have considered alternative structures for the Ni-A species including oxidation of thiolate ligands to either sulfenate or sulfenic acid. Here, we report both new and revised [NiFe]-hydrogenases structures and conclude, taking into account corresponding characterizations by Fourier transform infrared spectroscopy (FTIR), that the Ni-A species contains oxidized cysteine and bridging hydroxide ligands instead of the peroxide ligand we proposed earlier. Our analysis was rendered difficult by the typical formation of mixtures of unready oxidized states that, furthermore, can be reduced by X-ray induced photoelectrons. The present study could be carried out thanks to the use of Desulfovibrio fructosovorans [NiFe]-hydrogenase mutants with special properties. In addition to the Ni-A state, crystallographic results are also reported for two diamagnetic unready states, allowing the proposal of a revised oxidized inactive Ni-SU model and a new structure characterized by a persulfide ion that is assigned to an Ni-'Sox' species.
Assuntos
Proteínas de Bactérias/química , Hidrogenase/química , Methylophilaceae/enzimologia , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Domínio Catalítico , Cristalografia por Raios X , Hidrogenase/genética , Ferro/química , Modelos Moleculares , Níquel/química , Oxirredução , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
The class of [NiFe]-hydrogenases comprises oxygen-sensitive periplasmic (PH) and oxygen-tolerant membrane-bound (MBH) enzymes. For three PHs and four MBHs from six bacterial species, structural features of the nickel-iron active site of hydrogen turnover and of the iron-sulfur clusters functioning in electron transfer were determined using X-ray absorption spectroscopy (XAS). Fe-XAS indicated surplus oxidized iron and a lower number of ~2.7 Å Fe-Fe distances plus additional shorter and longer distances in the oxidized MBHs compared to the oxidized PHs. This supported a double-oxidized and modified proximal FeS cluster in all MBHs with an apparent trimer-plus-monomer arrangement of its four iron atoms, in agreement with crystal data showing a [4Fe3S] cluster instead of a [4Fe4S] cubane as in the PHs. Ni-XAS indicated coordination of the nickel by the thiol group sulfurs of four conserved cysteines and at least one iron-oxygen bond in both MBH and PH proteins. Structural differences of the oxidized inactive [NiFe] cofactor of MBHs in the Ni-B state compared to PHs in the Ni-A state included a ~0.05 Å longer Ni-O bond, a two times larger spread of the Ni-S bond lengths, and a ~0.1 Å shorter Ni-Fe distance. The modified proximal [4Fe3S] cluster, weaker binding of the Ni-Fe bridging oxygen species, and an altered localization of reduced oxygen species at the active site may each contribute to O2 tolerance.
Assuntos
Hidrogenase/química , Proteínas Ferro-Enxofre/química , Oxigênio/metabolismo , Espectroscopia por Absorção de Raios X/métodos , Sítios de Ligação , OxirreduçãoRESUMO
The genome of the facultative anaerobic γ-proteobacterium Shewanella oneidensis MR-1 encodes for three terminal oxidases: a bd-type quinol oxidase and two heme-copper oxidases, a A-type cytochrome c oxidase and a cbb 3-type oxidase. In this study, we used a biochemical approach and directly measured oxidase activities coupled to mass-spectrometry analysis to investigate the physiological role of the three terminal oxidases under aerobic and microaerobic conditions. Our data revealed that the cbb 3-type oxidase is the major terminal oxidase under aerobic conditions while both cbb 3-type and bd-type oxidases are involved in respiration at low-O2 tensions. On the contrary, the low O2-affinity A-type cytochrome c oxidase was not detected in our experimental conditions even under aerobic conditions and would therefore not be required for aerobic respiration in S. oneidensis MR-1. In addition, the deduced amino acid sequence suggests that the A-type cytochrome c oxidase is a ccaa 3-type oxidase since an uncommon extra-C terminal domain contains two c-type heme binding motifs. The particularity of the aerobic respiratory pathway and the physiological implication of the presence of a ccaa 3-type oxidase in S. oneidensis MR-1 are discussed.
Assuntos
Oxirredutases/metabolismo , Shewanella/metabolismo , Aerobiose , Membrana Celular/química , Membrana Celular/metabolismo , Respiração Celular/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Ativação Enzimática , Deleção de Genes , Ordem dos Genes , Família Multigênica , Oxirredutases/genética , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Shewanella/genéticaRESUMO
In this paper, the hydrogen (H2)-dependent discoloration of azo dye amaranth by Shewanella oneidensis MR-1 was investigated. Experiments with hydrogenase-deficient strains demonstrated that periplasmic [Ni-Fe] hydrogenase (HyaB) and periplasmic [Fe-Fe] hydrogenase (HydA) are both respiratory hydrogenases of dissimilatory azoreduction in S. oneidensis MR-1. These findings suggest that HyaB and HydA can function as uptake hydrogenases that couple the oxidation of H2 to the reduction of amaranth to sustain cellular growth. This constitutes to our knowledge the first report of the involvement of [Fe-Fe] hydrogenase in a bacterial azoreduction process. Assays with respiratory inhibitors indicated that a menaquinone pool and different cytochromes were involved in the azoreduction process. High-performance liquid chromatography analysis revealed that flavin mononucleotide and riboflavin were secreted in culture supernatant by S. oneidensis MR-1 under H2-dependent conditions with concentration of 1.4 and 2.4 µmol g protein(-1), respectively. These endogenous flavins were shown to significantly accelerate the reduction of amaranth at micromolar concentrations acting as electron shuttles between the cell surface and the extracellular azo dye. This work may facilitate a better understanding of the mechanisms of azoreduction by S. oneidensis MR-1 and may have practical applications for microbiological treatments of dye-polluted industrial effluents.
Assuntos
Corante Amaranto/metabolismo , Flavinas/metabolismo , Hidrogenase/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Shewanella/enzimologia , Shewanella/metabolismo , Amaranthus , Cromatografia Líquida de Alta Pressão , Elétrons , Oxirredução , Shewanella/crescimento & desenvolvimentoRESUMO
We studied the mechanism of aerobic inactivation of Desulfovibrio fructosovorans nickel-iron (NiFe) hydrogenase by quantitatively examining the results of electrochemistry, EPR and FTIR experiments. They suggest that, contrary to the commonly accepted mechanism, the attacking O(2) is not incorporated as an active site ligand but, rather, acts as an electron acceptor. Our findings offer new ways toward the understanding of O(2) inactivation and O(2) tolerance in NiFe hydrogenases.
Assuntos
Hidrogenase/metabolismo , Oxigênio/metabolismo , Desulfovibrio/enzimologia , Técnicas Eletroquímicas , Espectroscopia de Ressonância de Spin Eletrônica , Hidrogenase/química , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Nickel-containing hydrogenases, the biological catalysts of oxidation and production, reversibly inactivate under anaerobic, oxidizing conditions. We aim at understanding the mechanism of (in)activation and what determines its kinetics, because there is a correlation between fast reductive reactivation and oxygen tolerance, a property of some hydrogenases that is very desirable from the point of view of biotechnology. Direct electrochemistry is potentially very useful for learning about the redox-dependent conversions between active and inactive forms of hydrogenase, but the voltammetric signals are complex and often misread. Here we describe simple analytical models that we used to characterize and compare 16 mutants, obtained by substituting the position-74 valine of the -sensitive NiFe hydrogenase from Desulfovibrio fructosovorans. We observed that this substitution can accelerate reactivation up to 1,000-fold, depending on the polarity of the position 74 amino acid side chain. In terms of kinetics of anaerobic (in)activation and oxygen tolerance, the valine-to-histidine mutation has the most spectacular effect: The V74H mutant compares favorably with the -tolerant hydrogenase from Aquifex aeolicus, which we use here as a benchmark.
Assuntos
Biotecnologia/métodos , Desulfovibrio/enzimologia , Ativação Enzimática/genética , Hidrogenase/genética , Hidrogenase/metabolismo , Modelos Biológicos , Substituição de Aminoácidos/genética , Anaerobiose , Ativação Enzimática/fisiologia , Cinética , Mutação de Sentido Incorreto/genética , OxirreduçãoRESUMO
When enzymes are optimized for biotechnological purposes, the goal often is to increase stability or catalytic efficiency. However, many enzymes reversibly convert their substrate and product, and if one is interested in catalysis in only one direction, it may be necessary to prevent the reverse reaction. In other cases, reversibility may be advantageous because only an enzyme that can operate in both directions can turnover at a high rate even under conditions of low thermodynamic driving force. Therefore, understanding the basic mechanisms of reversibility in complex enzymes should help the rational engineering of these proteins. Here, we focus on NiFe hydrogenase, an enzyme that catalyzes H(2) oxidation and production, and we elucidate the mechanism that governs the catalytic bias (the ratio of maximal rates in the two directions). Unexpectedly, we found that this bias is not mainly determined by redox properties of the active site, but rather by steps which occur on sites of the proteins that are remote from the active site. We evidence a novel strategy for tuning the catalytic bias of an oxidoreductase, which consists in modulating the rate of a step that is limiting only in one direction of the reaction, without modifying the properties of the active site.
Assuntos
Desulfovibrio/enzimologia , Hidrogenase/metabolismo , Domínio Catalítico , Desulfovibrio/química , Desulfovibrio/genética , Hidrogenase/química , Hidrogenase/genética , Modelos Moleculares , Mutação , Oxirredução , TermodinâmicaRESUMO
We have investigated O2 and H2 transport across a NiFe hydrogenase at the atomic scale by means of computational methods. The Wild Type protein has been compared with the V74Q mutant. Two distinct methodologies have been applied to study the gas access to the active site. Temperature locally enhanced sampling simulations have emphasized the importance of protein dynamics on gas diffusion. The O2 diffusion free energy profiles, obtained by umbrella sampling, are in agreement with the known kinetic data and show that in the V74Q mutant, the inhibition process is lowered from both a kinetic and a thermodynamic point of view.
Assuntos
Desulfovibrio/enzimologia , Hidrogênio/metabolismo , Hidrogenase/metabolismo , Oxigênio/metabolismo , Domínio Catalítico , Desulfovibrio/química , Desulfovibrio/genética , Difusão , Hidrogênio/química , Hidrogenase/química , Hidrogenase/genética , Cinética , Modelos Moleculares , Oxigênio/química , Mutação Puntual , TermodinâmicaRESUMO
Electrons are transferred over long distances along chains of FeS clusters in hydrogenases, mitochondrial complexes, and many other respiratory enzymes. It is usually presumed that electron transfer is fast in these systems, despite the fact that there has been no direct measurement of rates of FeS-to-FeS electron transfer in any respiratory enzyme. In this context, we propose and apply to NiFe hydrogenase an original strategy that consists of quantitatively interpreting the variations of steady-state activity that result from changing the nature of the FeS clusters which connect the active site to the redox partner, and/or the nature of the redox partner. Rates of intra- and intermolecular electron transfer are deduced from such large data sets. The mutation-induced variations of electron transfer rates cannot be explained by changes in intercenter distances and reduction potentials. This establishes that FeS-to-FeS rate constants are extremely sensitive to the nature and coordination of the centers.
Assuntos
Hidrogenase/metabolismo , Desulfovibrio vulgaris/enzimologia , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Hidrogenase/química , Ferro/química , Cinética , Modelos Moleculares , Conformação Proteica , Enxofre/químicaRESUMO
Hydrogenases are efficient biological catalysts of H(2) oxidation and production. Most of them are inhibited by O(2), and a prerequisite for their use in biotechnological applications under air is to improve their oxygen tolerance. We have previously shown that exchanging the residue at position 74 in the large subunit of the oxygen-sensitive [NiFe] hydrogenase from Desulfovibrio fructosovorans could impact the reaction of the enzyme with O(2) (Dementin, S.; J. Am. Chem. Soc. 2009, 131, 10156-10164; Liebgott, P. P.; Nat. Chem. Biol. 2010, 6, 63-70). This residue, a valine in the wild-type enzyme, located at the bottleneck of the gas channel near the active site, has here been exchanged with a cysteine. A thorough characterization using a combination of kinetic, spectroscopic (EPR, FTIR), and electrochemical studies demonstrates that the V74C mutant has features of the naturally occurring oxygen-tolerant membrane-bound hydrogenases (MBH). The mutant is functional during several minutes under O(2), has impaired H(2)-production activity, and has a weaker affinity for CO than the WT. Upon exposure to O(2), it is converted into the more easily reactivatable inactive form, Ni-B, and this inactive state reactivates about 20 times faster than in the WT enzyme. Control experiments carried out with the V74S and V74N mutants indicate that protonation of the position 74 residue is not the reason the mutants reactivate faster than the WT enzyme. The electrochemical behavior of the V74C mutant toward O(2) is intermediate between that of the WT enzyme from D. fructosovorans and the oxygen-tolerant MBH from Aquifex aeolicus.
Assuntos
Domínio Catalítico , Cisteína , Hidrogenase/química , Hidrogenase/metabolismo , Mutação , Oxigênio/farmacologia , Valina , Aerobiose , Anaerobiose , Monóxido de Carbono/farmacologia , Membrana Celular/metabolismo , Desulfovibrio/enzimologia , Medição da Troca de Deutério , Eletroquímica , Ativação Enzimática/efeitos dos fármacos , Bactérias Gram-Negativas/enzimologia , Hidrogênio/metabolismo , Hidrogenase/antagonistas & inibidores , Hidrogenase/genética , Cinética , Modelos Moleculares , Oxirredução , Análise Espectral , TermodinâmicaRESUMO
In hydrogenases and many other redox enzymes, the buried active site is connected to the solvent by a molecular channel whose structure may determine the enzyme's selectivity with respect to substrate and inhibitors. The role of these channels has been addressed using crystallography and molecular dynamics, but kinetic data are scarce. Using protein film voltammetry, we determined and then compared the rates of inhibition by CO and O2 in ten NiFe hydrogenase mutants and two FeFe hydrogenases. We found that the rate of inhibition by CO is a good proxy of the rate of diffusion of O2 toward the active site. Modifying amino acids whose side chains point inside the tunnel can slow this rate by orders of magnitude. We quantitatively define the relations between diffusion, the Michaelis constant for H2 and rates of inhibition, and we demonstrate that certain enzymes are slowly inactivated by O2 because access to the active site is slow.
Assuntos
Desulfovibrio/enzimologia , Hidrogenase/química , Oxigênio/química , Aminoácidos/química , Monóxido de Carbono/química , Domínio Catalítico , Cristalografia por Raios X/métodos , Difusão , Eletroquímica/métodos , Espectroscopia de Ressonância de Spin Eletrônica , Hidrogênio/química , Cinética , Modelos Moleculares , Conformação Molecular , Simulação de Dinâmica MolecularRESUMO
Hydrogenases catalyze the conversion between 2H(+) + 2e(-) and H(2)(1). Most of these enzymes are inhibited by O(2), which represents a major drawback for their use in biotechnological applications. Improving hydrogenase O(2) tolerance is therefore a major contemporary challenge to allow the implementation of a sustainable hydrogen economy. We succeeded in improving O(2) tolerance, which we define here as the ability of the enzyme to resist for several minutes to O(2) exposure, by substituting with methionines small hydrophobic residues strongly conserved in the gas channel. Remarkably, the mutated enzymes remained active in the presence of an O(2) concentration close to that found in aerobic solutions in equilibrium with air, while the wild type enzyme is inhibited in a few seconds. Crystallographic and spectroscopic studies showed that the structure and the chemistry at the active site are not affected by the mutations. Kinetic studies demonstrated that the inactivation is slower and reactivation faster in these mutants. We propose that in addition to restricting O(2) diffusion to the active site of the enzyme, methionine may also interact with bound peroxide and provide an assisted escape route for H(2)O(2) toward the gas channel. These results show for the first time that it is possible to improve O(2)-tolerance of [NiFe] hydrogenases, making possible the development of biohydrogen production systems.
Assuntos
Hidrogenase/metabolismo , Metionina/metabolismo , Oxigênio/metabolismo , Domínio Catalítico , Difusão , Gases/química , Gases/metabolismo , Hidrogenase/química , Metionina/química , Oxigênio/químicaRESUMO
Chronoamperometric experiments with adsorbed electrocatalysts are commonly performed either for analytical purposes or for studying the catalytic mechanism of a redox enzyme. In the context of amperometric sensors, the current may be recorded as a function of time while the analyte concentration is being increased to determine a linearity range. In mechanistic studies of redox enzymes, chronoamperometry proved powerful for untangling the effects of electrode potential and time, which are convoluted in cyclic voltammetric measurements, and for studying the energetics and kinetics of inhibition. In all such experiments, the fact that the catalyst's coverage and/or activity decreases over time distorts the data. This may hide meaningful features, introduce systematic errors, and limit the accuracy of the measurements. We propose a general and surprisingly simple method for correcting for electrocatalyst desorption and inactivation, which greatly increases the precision of chronoamperometric experiments. Rather than subtracting a baseline, this consists in dividing the current, either by a synthetic signal that is proportional to the instant electroactive coverage or by the signal recorded in a control experiment. In the latter, the change in current may result from film loss only or from film loss plus catalyst inactivation. We describe the different strategies for obtaining the control signal by analyzing various data recorded with adsorbed redox enzymes: nitrate reductase, NiFe hydrogenase, and FeFe hydrogenase. In each case we discuss the trustfulness and the benefit of the correction. This method also applies to experiments where electron transfer is mediated, rather than direct, providing the current is proportional to the time-dependent concentration of catalyst.
Assuntos
Artefatos , Biocatálise , Técnicas Eletroquímicas/métodos , Adsorção , Condutividade Elétrica , Enzimas/química , Enzimas/metabolismo , SoftwareRESUMO
BACKGROUND: The eukaryotic green alga, Chlamydomonas reinhardtii, produces H2 under anaerobic conditions, in a reaction catalysed by a [Fe-Fe] hydrogenase HydA1. For further biochemical and biophysical studies a suitable expression system of this enzyme should be found to overcome its weak expression in the host organism. Two heterologous expression systems used up to now have several advantages. However they are not free from some drawbacks. In this work we use bacterium Shewanella oneidensis as a new and efficient system for expression and maturation of HydA1 from Chlamydomonas reinhardtii. RESULTS: Based on codon usage bias and hydrogenase maturation ability, the bacterium S. oneidensis, which possesses putative [Fe-Fe] and [Ni-Fe] hydrogenase operons, was selected as the best potential host for C. reinhardtii [Fe-Fe] hydrogenase expression. Hydrogen formation by S. oneidensis strain AS52 (Delta hydA Delta hyaB) transformed with a plasmid bearing CrHydA1 and grown in the presence of six different substrates for anaerobic respiration was determined. A significant increase in hydrogen evolution was observed for cells grown in the presence of trimethylamine oxide, dimethylsulfoxide and disodium thiosulfate, showing that the system of S. oneidensis is efficient for heterologous expression of algal [Fe-Fe] hydrogenase. CONCLUSION: In the present work a new efficient system for heterologous expression and maturation of C. reinhardtii hydrogenase has been developed. HydA1 of C. reinhardtii was purified and shown to contain 6 Fe atoms/molecule of protein, as expected. Using DMSO, TMAO or thiosulfate as substrates for anaerobic respiration during the cell growth, 0.4 - 0.5 mg l(-1)(OD600 = 1) of catalytically active HydA1 was obtained with hydrogen evolution rate of approximately 700 micromol H2 mg(-1) min(-1).
Assuntos
Chlamydomonas reinhardtii/enzimologia , Chlamydomonas reinhardtii/genética , Hidrogenase/química , Hidrogenase/metabolismo , Engenharia de Proteínas/métodos , Shewanella/enzimologia , Shewanella/genética , Animais , Ativação Enzimática , Estabilidade Enzimática , Regulação Enzimológica da Expressão Gênica/fisiologia , Hidrogenase/genética , Hidrogenase/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Hydrogenases, which catalyze H(2) to H(+) conversion as part of the bioenergetic metabolism of many microorganisms, are among the metalloenzymes for which a gas-substrate tunnel has been described by using crystallography and molecular dynamics. However, the correlation between protein structure and gas-diffusion kinetics is unexplored. Here, we introduce two quantitative methods for probing the rates of diffusion within hydrogenases. One uses protein film voltammetry to resolve the kinetics of binding and release of the competitive inhibitor CO; the other is based on interpreting the yield in the isotope exchange assay. We study structurally characterized mutants of a NiFe hydrogenase, and we show that two mutations, which significantly narrow the tunnel near the entrance of the catalytic center, decrease the rates of diffusion of CO and H(2) toward and from the active site by up to 2 orders of magnitude. This proves the existence of a functional channel, which matches the hydrophobic cavity found in the crystal. However, the changes in diffusion rates do not fully correlate with the obstruction induced by the mutation and deduced from the x-ray structures. Our results demonstrate the necessity of measuring diffusion rates and emphasize the role of side-chain dynamics in determining these.
Assuntos
Monóxido de Carbono/química , Desulfovibrio/enzimologia , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Hidrogênio/química , Hidrogenase/química , Sítios de Ligação/genética , Cristalografia por Raios X , Desulfovibrio/genética , Eletroquímica , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Gases/química , Hidrogenase/antagonistas & inibidores , Hidrogenase/genética , Interações Hidrofóbicas e Hidrofílicas , Cinética , Mutação , Ligação Proteica/genética , Estrutura Terciária de Proteína/genéticaAssuntos
Biomassa , Eletroquímica , Hidrogenase/antagonistas & inibidores , Hidrogenase/metabolismo , Análise Espectral , Anaerobiose , Sítios de Ligação , Monóxido de Carbono/farmacologia , Catálise , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Hidrogênio/química , Hidrogênio/metabolismo , Hidrogenase/química , Proteínas Ferro-Enxofre/antagonistas & inibidores , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , Modelos Químicos , Oxirredução , Oxigênio/química , Oxigênio/metabolismoRESUMO
In NiFe hydrogenases, electrons are transferred from the active site to the redox partner via a chain of three Iron-Sulfur clusters, and the surface-exposed [4Fe4S] cluster has an unusual His(Cys)3 ligation. When this Histidine (H184 in Desulfovibrio fructosovorans) is changed into a cysteine or a glycine, a distal cubane is still assembled but the oxidative activity of the mutants is only 1.5 and 3% of that of the WT, respectively. We compared the activities of the WT and engineered enzymes for H2 oxidation, H+ reduction and H/D exchange, under various conditions: (i) either with the enzyme directly adsorbed onto an electrode or using soluble redox partners, and (ii) in the presence of exogenous ligands whose binding to the exposed Fe of H184G was expected to modulate the properties of the distal cluster. Protein film voltammetry proved particularly useful to unravel the effects of the mutations on inter and intramolecular electron transfer (ET). We demonstrate that changing the coordination of the distal cluster has no effect on cluster assembly, protein stability, active-site chemistry and proton transfer; however, it slows down the first-order rates of ET to and from the cluster. All-sulfur coordination is actually detrimental to ET, and intramolecular (uphill) ET is rate determining in the glycine variant. This demonstrates that although [4Fe4S] clusters are robust chemical constructs, the direct protein ligands play an essential role in imparting their ability to transfer electrons.
Assuntos
Hidrogenase/química , Hidrogenase/metabolismo , Proteínas Ferro-Enxofre/química , Proteínas Ferro-Enxofre/metabolismo , Desulfovibrio/enzimologia , Desulfovibrio/genética , Eletroquímica , Espectroscopia de Ressonância de Spin Eletrônica , Concentração de Íons de Hidrogênio , Hidrogenase/antagonistas & inibidores , Hidrogenase/genética , Imidazóis/química , Imidazóis/metabolismo , Proteínas Ferro-Enxofre/genética , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , SoluçõesRESUMO
The gene encoding Desulfovibrio gigas flavoredoxin was deleted to elucidate its physiological role in the sulfate metabolism. Disruption of flr gene strongly inhibited the reduction of thiosulfate and exhibited a reduced growth in the presence of sulfite with lactate as electron donor. The growth with sulfate was not however affected by the lack of this protein. Additionally, flr mutant cells revealed a decrease of about 50% in the H2 consumption rate using thiosulfate as electron acceptor. Altogether, our results show in vivo that during sulfite respiration, trithionate and thiosulfate are produced and that flavoredoxin is specific for thiosulfate reduction.