RESUMEN
The observation that some homologous enzymes have the same active site but very different catalytic properties demonstrates the importance of long-range effects in enzyme catalysis, but these effects are often difficult to rationalize. The NiFe hydrogenases 1 and 2 (Hyd 1 and Hyd 2) from E. coli both consist of a large catalytic subunit that embeds the same dinuclear active site and a small electron-transfer subunit with a chain of three FeS clusters. Hyd 1 is mostly active in H2 oxidation and resistant to inhibitors, whereas Hyd 2 also catalyzes H2 production and is strongly inhibited by O2 and CO. Based on structural and site-directed mutagenesis data, it is currently believed that the catalytic bias and tolerance to O2 of Hyd 1 are defined by the distal and proximal FeS clusters, respectively. To test these hypotheses, we produced and characterized a hybrid enzyme made of the catalytic subunit of Hyd 1 and the electron transfer subunit of Hyd 2. We conclude that catalytic bias and sensitivity to CO are set by the catalytic subunit rather than by the electron transfer chain. We confirm the importance of the proximal cluster in making the enzyme Hyd 1 resist long-term exposure to O2, but we show that other structural determinants, in both subunits, contribute to O2 tolerance. A similar strategy based on the design of chimeric heterodimers could be used in the future to elucidate various structure-function relationships in hydrogenases and other multimeric metalloenzymes and to engineer useful hydrogenases that combine the desirable properties of distinct, homologous enzymes.
Asunto(s)
Electrones , Escherichia coli , Escherichia coli/genética , Catálisis , OxígenoRESUMEN
Desulfovibrio fructosovorans, a sulfate-reducing bacterium, possesses six gene clusters encoding six hydrogenases catalyzing the reversible oxidation of H2 into protons and electrons. Among them, Hnd is an electron-bifurcating hydrogenase, coupling the exergonic reduction of NAD+ to the endergonic reduction of a ferredoxin with electrons derived from H2 . It was previously hypothesized that its biological function involves the production of NADPH necessary for biosynthetic purposes. However, it was subsequently demonstrated that Hnd is instead a NAD+ -reducing enzyme, thus its specific function has yet to be established. To understand the physiological role of Hnd in D. fructosovorans, we compared the hnd deletion mutant with the wild-type strain grown on pyruvate. Growth, metabolite production and consumption, and gene expression were compared under three different growth conditions. Our results indicate that hnd is strongly regulated at the transcriptional level and that its deletion has a drastic effect on the expression of genes for two enzymes, an aldehyde ferredoxin oxidoreductase and an alcohol dehydrogenase. We demonstrated here that Hnd is involved in ethanol metabolism when bacteria grow fermentatively and proposed that Hnd might oxidize part of the H2 produced during fermentation generating both NADH and reduced ferredoxin for ethanol production via its electron bifurcation mechanism.
Asunto(s)
Hidrogenasas , Desulfovibrio , Electrones , Etanol , Ferredoxinas/metabolismo , Hidrógeno/metabolismo , Hidrogenasas/genética , Hidrogenasas/metabolismo , NAD/metabolismo , Oxidación-Reducción , Ácido PirúvicoRESUMEN
BACKGROUND: The ability of some photosynthetic microorganisms, particularly cyanobacteria and microalgae, to produce hydrogen (H2) is a promising alternative for renewable, clean-energy production. However, the most recent, related studies point out that much improvement is needed for sustainable cyanobacterial-based H2 production to become economically viable. In this study, we investigated the impact of induced O2-consumption on H2 photoproduction yields in the heterocyte-forming, N2-fixing cyanobacterium Nostoc PCC7120. RESULTS: The flv3B gene, encoding a flavodiiron protein naturally expressed in Nostoc heterocytes, was overexpressed. Under aerobic and phototrophic growth conditions, the recombinant strain displayed a significantly higher H2 production than the wild type. Nitrogenase activity assays indicated that flv3B overexpression did not enhance the nitrogen fixation rates. Interestingly, the transcription of the hox genes, encoding the NiFe Hox hydrogenase, was significantly elevated, as shown by the quantitative RT-PCR analyses. CONCLUSION: We conclude that the overproduced Flv3B protein might have enhanced O2-consumption, thus creating conditions inducing hox genes and facilitating H2 production. The present study clearly demonstrates the potential to use metabolic engineered cyanobacteria for photosynthesis driven H2 production.
Asunto(s)
Proteínas Bacterianas/metabolismo , Hidrógeno/metabolismo , Nostoc/metabolismo , Oxígeno/metabolismo , Proteínas Bacterianas/genética , Genes Homeobox , Hidrogenasas/genética , Hidrogenasas/metabolismo , Ingeniería Metabólica , Nitrógeno/metabolismo , Fijación del Nitrógeno , Nostoc/genética , FotosíntesisRESUMEN
Strictly anaerobic bacteria of the Clostridium genus have attracted great interest as potential cell factories for molecular hydrogen production purposes. In addition to being a useful approach to this process, dark fermentation has the advantage of using the degradation of cheap agricultural residues and industrial wastes for molecular hydrogen production. However, many improvements are still required before large-scale hydrogen production from clostridial metabolism is possible. Here we review the literature on the basic biological processes involved in clostridial hydrogen production, and present the main advances obtained so far in order to enhance the hydrogen productivity, as well as suggesting some possible future prospects.
Asunto(s)
Clostridium/enzimología , Clostridium/metabolismo , Hidrógeno/metabolismo , Anaerobiosis , Fermentación , Residuos IndustrialesRESUMEN
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.
Asunto(s)
Clostridium acetobutylicum/enzimología , Hidrógeno/metabolismo , Hidrogenasas/genética , Hidrogenasas/metabolismo , Microbiología Industrial/métodos , Nostoc/genética , Organismos Modificados Genéticamente/genética , Organismos Modificados Genéticamente/metabolismoRESUMEN
The extremely acidophilic archaeon Ferroplasma acidiphilum is found in iron-rich biomining environments and is an important micro-organism in naturally occurring microbial communities in acid mine drainage. F. acidiphilum is an iron oxidizer that belongs to the order Thermoplasmatales (Euryarchaeota), which harbors the most extremely acidophilic micro-organisms known so far. At present, little is known about the nature or the structural and functional organization of the proteins in F. acidiphilum that impact the iron biogeochemical cycle. We combine here biochemical and biophysical techniques such as enzyme purification, activity measurements, proteomics and spectroscopy to characterize the iron oxidation pathway(s) in F. acidiphilum. We isolated two respiratory membrane protein complexes: a 850 kDa complex containing an aa3-type cytochrome oxidase and a blue copper protein, which directly oxidizes ferrous iron and reduces molecular oxygen, and a 150 kDa cytochrome ba complex likely composed of a di-heme cytochrome and a Rieske protein. We tentatively propose that both of these complexes are involved in iron oxidation respiratory chains, functioning in the so-called uphill and downhill electron flow pathways, consistent with autotrophic life. The cytochrome ba complex could possibly play a role in regenerating reducing equivalents by a reverse ('uphill') electron flow. This study constitutes the first detailed biochemical investigation of the metalloproteins that are potentially directly involved in iron-mediated energy conservation in a member of the acidophilic archaea of the genus Ferroplasma.
Asunto(s)
Proteínas Arqueales/metabolismo , Membrana Celular/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Compuestos Ferrosos/química , Complejos Multiproteicos/metabolismo , Oxígeno/metabolismo , Thermoplasmales/clasificación , Ácidos/química , Aerobiosis/fisiología , Proteínas Arqueales/química , Membrana Celular/química , Transporte de Electrón , Complejo IV de Transporte de Electrones/química , Compuestos Ferrosos/metabolismo , Complejos Multiproteicos/química , Operón , Oxidación-Reducción , Thermoplasmales/crecimiento & desarrollo , Thermoplasmales/metabolismoRESUMEN
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.
Asunto(s)
Colorante de Amaranto/metabolismo , Flavinas/metabolismo , Hidrogenasas/metabolismo , Proteínas Hierro-Azufre/metabolismo , Shewanella/enzimología , Shewanella/metabolismo , Amaranthus , Cromatografía Líquida de Alta Presión , Electrones , Oxidación-Reducción , Shewanella/crecimiento & desarrolloRESUMEN
[This corrects the article DOI: 10.1016/j.bbadva.2023.100090.].
RESUMEN
Solidesulfovibrio fructosivorans (formely Desulfovibrio fructosovorans), an anaerobic sulfate-reducing bacterium, possesses six gene clusters encoding six hydrogenases catalyzing the reversible oxidation of hydrogen gas (H2) into protons and electrons. One of these, named Hnd, was demonstrated to be an electron-bifurcating hydrogenase Hnd (Kpebe et al., 2018). It couples the exergonic reduction of NAD+ to the endergonic reduction of a ferredoxin with electrons derived from H2 and whose function has been recently shown to be involved in ethanol production under pyruvate fermentation (Payne 2022). To understand further the physiological role of Hnd in S. fructosivorans, we compared the mutant deleted of part of the hnd gene with the wild-type strain grown on pyruvate without sulfate using NMR-based metabolomics. Our results confirm that Hnd is profoundly involved in ethanol metabolism, but also indirectly intervenes in global carbon metabolism and additional metabolic processes such as the biosynthesis of branched-chain amino acids. We also highlight the metabolic reprogramming induced by the deletion of hndD that leads to the upregulation of several NADP-dependent pathways.
Asunto(s)
Hidrogenasas , Electrones , Fermentación , Hidrógeno/metabolismo , Hidrogenasas/genética , Hidrogenasas/química , Hidrogenasas/metabolismo , Oxidación-Reducción , Ácido Pirúvico , Desulfovibrionaceae/química , Desulfovibrionaceae/metabolismoRESUMEN
The tetrameric cytoplasmic FeFe hydrogenase Hnd from Solidesulfovibrio fructosivorans (formely Desulfovibrio fructosovorans) catalyses H2 oxidation and couples the exergonic reduction of NAD+ to the endergonic reduction of a ferredoxin by using a flavin-based electron-bifurcating mechanism. Regarding its implication in the bacterial physiology, we previously showed that Hnd, which is non-essential when bacteria grow fermentatively on pyruvate, is involved in ethanol metabolism. Under these conditions, it consumes H2 to produce reducing equivalents for ethanol production as a fermentative product. In this study, the approach implemented was to compare the two S. fructosivorans WT and the hndD deletion mutant strains when grown on ethanol as the sole carbon and energy source. Based on the determination of bacterial growth, metabolite consumption and production, gene expression followed by RT-q-PCR, and Hnd protein level followed by mass spectrometry, our results confirm the role of Hnd hydrogenase in the ethanol metabolism and furthermore uncover for the first time an essential function for a Desulfovibrio hydrogenase. Hnd is unequivocally required for S. fructosivorans growth on ethanol, and we propose that it produces H2 from NADH and reduced ferredoxin generated by an alcohol dehydrogenase and an aldehyde ferredoxin oxidoreductase catalyzing the conversion of ethanol into acetate. The produced H2 could then be recycled and used for sulfate reduction. Hnd is thus a reversible hydrogenase that operates in H2-consumption by an electron-bifurcating mechanism during pyruvate fermentation and in H2-production by an electron-confurcating mechanism when the bacterium uses ethanol as electron donor.
RESUMEN
Protein Film Electrochemistry is a technique in which a redox enzyme is directly wired to an electrode, which substitutes for the natural redox partner. In this technique, the electrical current flowing through the electrode is proportional to the catalytic activity of the enzyme. However, in most cases, the amount of enzyme molecules contributing to the current is unknown and the absolute turnover frequency cannot be determined. Here, we observe the formation of electrocatalytically active films of E. coli hydrogenase 1 by rotating an electrode in a sub-nanomolar solution of enzyme. This process is slow, and we show that it is mass-transport limited. Measuring the rate of the immobilization allows the determination of an estimation of the turnover rate of the enzyme, which appears to be much greater than that deduced from solution assays under the same conditions.
RESUMEN
Aquifex aeolicus, a hyperthermophilic and microaerophilic bacterium, obtains energy for growth from inorganic compounds alone. It was previously proposed that one of the respiratory pathways in this organism consists of the electron transfer from hydrogen sulfide (H(2)S) to molecular oxygen. H(2)S is oxidized by the sulfide quinone reductase, a membrane-bound flavoenzyme, which reduces the quinone pool. We have purified and characterized a novel membrane-bound multienzyme supercomplex that brings together all the molecular components involved in this bioenergetic chain. Our results indicate that this purified structure consists of one dimeric bc(1) complex (complex III), one cytochrome c oxidase (complex IV), and one or two sulfide quinone reductases as well as traces of the monoheme cytochrome c(555) and quinone molecules. In addition, this work strongly suggests that the cytochrome c oxidase in the supercomplex is a ba(3)-type enzyme. The supercomplex has a molecular mass of about 350 kDa and is enzymatically functional, reducing O(2) in the presence of the electron donor, H(2)S. This is the first demonstration of the existence of such a respirasome carrying a sulfide oxidase-oxygen reductase activity. Moreover, the kinetic properties of the sulfide quinone reductase change slightly when integrated in the supercomplex, compared with the free enzyme. We previously purified a complete respirasome involved in hydrogen oxidation and sulfur reduction from Aquifex aeolicus. Thus, two different bioenergetic pathways (sulfur reduction and sulfur oxidation) are organized in this bacterium as supramolecular structures in the membrane. A model for the energetic sulfur metabolism of Aquifex aeolicus is proposed.
Asunto(s)
Bacterias/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/química , Oxígeno/química , Proteínas Bacterianas/química , Espectroscopía de Resonancia por Spin del Electrón , Transporte de Electrón , Complejo IV de Transporte de Electrones/química , Hidrógeno/química , Sulfuro de Hidrógeno/química , Oxidación-Reducción , Consumo de Oxígeno , Espectrometría de Fluorescencia/métodos , Espectrofotometría Ultravioleta/métodosRESUMEN
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.
Asunto(s)
Dominio Catalítico , Cisteína , Hidrogenasas/química , Hidrogenasas/metabolismo , Mutación , Oxígeno/farmacología , Valina , Aerobiosis , Anaerobiosis , Monóxido de Carbono/farmacología , Membrana Celular/metabolismo , Desulfovibrio/enzimología , Medición de Intercambio de Deuterio , Electroquímica , Activación Enzimática/efectos de los fármacos , Bacterias Gramnegativas/enzimología , Hidrógeno/metabolismo , Hidrogenasas/antagonistas & inhibidores , Hidrogenasas/genética , Cinética , Modelos Moleculares , Oxidación-Reducción , Análisis Espectral , TermodinámicaRESUMEN
The concomitant presence of two distinctive polypeptide modules, which we have chosen to denominate as the "Y-junction" and the "flavin" module, is observed in 3D structures of enzymes as functionally diverse as complex I, NAD(P)-dependent [NiFe]-hydrogenases and NAD(P)-dependent formate dehydrogenases. Amino acid sequence conservation furthermore suggests that both modules are also part of NAD(P)-dependent [FeFe]-hydrogenases for which no 3D structure model is available yet. The flavin module harbours the site of interaction with the substrate NAD(P) which exchanges two electrons with a strictly conserved flavin moiety. The Y-junction module typically contains four iron-sulphur centres arranged to form a Y-shaped electron transfer conduit and mediates electron transfer between the flavin module and the catalytic units of the respective enzymes. The Y-junction module represents an electron transfer hub with three potential electron entry/exit sites. The pattern of specific redox centres present both in the Y-junction and the flavin module is correlated to present knowledge of these enzymes' functional properties. We have searched publicly accessible genomes for gene clusters containing both the Y-junction and the flavin module to assemble a comprehensive picture of the diversity of enzymes harbouring this dyad of modules and to reconstruct their phylogenetic relationships. These analyses indicate the presence of the dyad already in the last universal common ancestor and the emergence of complex I's EFG-module out of a subgroup of NAD(P)- dependent formate dehydrogenases.
Asunto(s)
Proteínas Bacterianas/metabolismo , Electrones , Flavinas/metabolismo , Formiato Deshidrogenasas/metabolismo , Hidrogenasas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Transporte de Electrón , Flavinas/química , Formiato Deshidrogenasas/química , Formiato Deshidrogenasas/genética , Hidrogenasas/química , Hidrogenasas/genética , FilogeniaRESUMEN
Hnd, an FeFe hydrogenase from Desulfovibrio fructosovorans, is a tetrameric enzyme that can perform flavin-based electron bifurcation. It couples the oxidation of H2 to both the exergonic reduction of NAD+ and the endergonic reduction of a ferredoxin. We previously showed that Hnd retains activity even when purified aerobically unlike other electron-bifurcating hydrogenases. In this study, we describe the purification of the enzyme under O2-free atmosphere and its biochemical and electrochemical characterization. Despite its complexity due to its multimeric composition, Hnd can catalytically and directly exchange electrons with an electrode. We characterized the catalytic and inhibition properties of this electron-bifurcating hydrogenase using protein film electrochemistry of Hnd by purifying Hnd aerobically or anaerobically, then comparing the electrochemical properties of the enzyme purified under the two conditions via protein film electrochemistry. Hydrogenases are usually inactivated under oxidizing conditions in the absence of dioxygen and can then be reactivated, to some extent, under reducing conditions. We demonstrate that the kinetics of this high potential inactivation/reactivation for Hnd show original properties: it depends on the enzyme purification conditions and varies with time, suggesting the coexistence and the interconversion of two forms of the enzyme. We also show that Hnd catalytic properties (Km for H2, diffusion and reaction at the active site of CO and O2) are comparable to those of standard hydrogenases (those which cannot catalyze electron bifurcation). These results suggest that the presence of the additional subunits, needed for electron bifurcation, changes neither the catalytic behavior at the active site, nor the gas diffusion kinetics but induces unusual rates of high potential inactivation/reactivation.
RESUMEN
The electrochemistry of membrane-bound [NiFe] hydrogenase I ([NiFe]-hase I) from the hyperthermophilic bacterium Aquifex aeolicus was investigated at gold and graphite electrodes. Direct and mediated H(2) oxidation were proved to be efficient in a temperature range of 25-70 degrees C, describing a potential window for H(2) oxidation similar to that of O(2)-tolerant hydrogenases. Search for enhancement of current densities and enzyme stability was achieved by the use of carbon nanotube coatings. We report high catalytic currents for H(2) oxidation up to 1 mA cm(-2), 10 times higher than at the bare electrode. Interestingly, high stability of the direct catalytic process was observed when encapsulating A. aeolicus [NiFe]-hase I into a carboxylic functionalized single walled carbon nanotube network. This suggests a peculiar interaction between the enzyme and the electrode material. The parameters that governed the orientation of the enzyme before electron transfer were thus investigated using self-assembled-monolayer gold electrodes. No control of the orientation by the charge or the hydrophobicity of the interface was demonstrated. This behavior was explained on the basis of a structural comparison between A. aeolicus [NiFe]-hase I and Desulfovibrio fructosovorans [NiFe] hydrogenase, which revealed the absence of acidic residues and an additional loop in the environment of the [4Fe-4S] distal cluster in A. aeolicus [NiFe]-hase I. Finally, the effect of inhibitors on the direct oxidation of H(2) by A. aeolicus [NiFe]-hase I encapsulated in a single walled carbon nanotube network was investigated. No inhibition by CO and tolerance toward O(2) were observed. Discussion of the reasons for such tolerance was undertaken on the basis of structural comparison with hydrogenases from aerobic bacteria.
Asunto(s)
Bacterias/enzimología , Electrodos , Enzimas Inmovilizadas , Oro/química , Hidrogenasas , Nanotubos de Carbono/química , Adsorción , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Técnicas Electroquímicas , Inhibidores Enzimáticos/metabolismo , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Hidrogenasas/química , Hidrogenasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Conformación Proteica , Alineación de SecuenciaRESUMEN
Hydrogen metabolism plays a central role in sulfate-reducing bacteria of the Desulfovibrio genus and is based on hydrogenases that catalyze the reversible conversion of protons into dihydrogen. These metabolically versatile microorganisms possess a complex hydrogenase system composed of several enzymes of both [FeFe]- and [NiFe]-type that can vary considerably from one Desulfovibrio species to another. This review covers the molecular and physiological aspects of hydrogenases and H2 metabolism in Desulfovibrio but focuses particularly on our model bacterium Desulfovibrio fructosovorans. The search of hydrogenase genes in more than 30 sequenced genomes provides an overview of the distribution of these enzymes in Desulfovibrio. Our discussion will consider the significance of the involvement of electron-bifurcation in H2 metabolism.
Asunto(s)
Proteínas Bacterianas/fisiología , Desulfovibrio/metabolismo , Hidrógeno/metabolismo , Hidrogenasas/fisiología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biocatálisis , Desulfovibrio/enzimología , Desulfovibrio/genética , Electrones , Regulación Bacteriana de la Expresión Génica , Variación Genética , Hidrogenasas/química , Hidrogenasas/genética , Hidrogenasas/metabolismo , Modelos BiológicosRESUMEN
Electron bifurcation is here described as a special case of the continuum of electron transfer reactions accessible to two-electron redox compounds with redox cooperativity. We argue that electron bifurcation is foremost an electrochemical phenomenon based on (a) strongly inverted redox potentials of the individual redox transitions, (b) a high endergonicity of the first redox transition, and (c) an escapement-type mechanism rendering completion of the first electron transfer contingent on occurrence of the second one. This mechanism is proposed to govern both the traditional quinone-based and the newly discovered flavin-based versions of electron bifurcation. Conserved and variable aspects of the spatial arrangement of electron transfer partners in flavoenzymes are assayed by comparing the presently available 3D structures. A wide sample of flavoenzymes is analyzed with respect to conserved structural modules and three major structural groups are identified which serve as basic frames for the evolutionary construction of a plethora of flavin-containing redox enzymes. We argue that flavin-based and other types of electron bifurcation are of primordial importance to free energy conversion, the quintessential foundation of life, and discuss a plausible evolutionary ancestry of the mechanism.
RESUMEN
The genome of the sulfate-reducing and anaerobic bacterium Desulfovibrio fructosovorans encodes different hydrogenases. Among them is Hnd, a tetrameric cytoplasmic [FeFe] hydrogenase that has previously been described as an NADP-specific enzyme (Malki et al., 1995). In this study, we purified and characterized a recombinant Strep-tagged form of Hnd and demonstrated that it is an electron-bifurcating enzyme. Flavin-based electron-bifurcation is a mechanism that couples an exergonic redox reaction to an endergonic one allowing energy conservation in anaerobic microorganisms. One of the three ferredoxins of the bacterium, that was named FdxB, was also purified and characterized. It contains a low-potential (Emâ¯=â¯-450â¯mV) [4Fe4S] cluster. We found that Hnd was not able to reduce NADP+, and that it catalyzes the simultaneous reduction of FdxB and NAD+. Moreover, Hnd is the first electron-bifurcating hydrogenase that retains activity when purified aerobically due to formation of an inactive state of its catalytic site protecting against O2 damage (Hinact). Hnd is highly active with the artificial redox partner (methyl viologen) and can perform the electron-bifurcation reaction to oxidize H2 with a specific activity of 10⯵mol of NADH/min/mg of enzyme. Surprisingly, the ratio between NADH and reduced FdxB varies over the reaction with a decreasing amount of FdxB reduced per NADH produced, indicating a more complex mechanism than previously described. We proposed a new mechanistic model in which the ferredoxin is recycled at the hydrogenase catalytic subunit.
Asunto(s)
Desulfovibrio/enzimología , Electrones , Hidrogenasas/metabolismo , Modelos Biológicos , Oxígeno/metabolismo , Secuencia de Aminoácidos , Biocatálisis , Desulfovibrio/genética , Ferredoxinas/genética , Ferredoxinas/metabolismo , Hidrogenasas/química , Hidrogenasas/genética , NAD/metabolismo , Espectrofotometría Ultravioleta , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
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.