A Chimeric NiFe Hydrogenase Heterodimer to Assess the Role of the Electron Transfer Chain in Tuning the Enzyme's Catalytic Bias and Oxygen Tolerance.

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.

Transport limited adsorption experiments give a new lower estimate of the turnover frequency of Escherichia coli hydrogenase 1.

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.

An essential role of the reversible electron-bifurcating hydrogenase Hnd for ethanol oxidation in Solidesulfovibrio fructosivorans.

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.

Carbon Fixation in the Chemolithoautotrophic Bacterium Aquifex aeolicus Involves Two Low-Potential Ferredoxins as Partners of the PFOR and OGOR Enzymes.

Aquifex aeolicus is a microaerophilic hydrogen- and sulfur -oxidizing bacterium that assimilates CO2 via the reverse tricarboxylic acid cycle (rTCA). Key enzymes of this pathway are pyruvate:ferredoxin oxidoreductase (PFOR) and 2-oxoglutarate:ferredoxin oxidoreductase (OGOR), which are responsible, respectively, for the reductive carboxylation of acetyl-CoA to pyruvate and of succinyl-CoA to 2-oxoglutarate, two energetically unfavorable reactions that require a strong reduction potential. We have confirmed, by biochemistry and proteomics, that A. aeolicus possesses a pentameric version of these enzyme complexes ((αßγδε)2) and that they are highly abundant in the cell. In addition, we have purified and characterized, from the soluble fraction of A. aeolicus, two low redox potential and oxygen-stable [4Fe-4S] ferredoxins (Fd6 and Fd7, E0 = -440 and -460 mV, respectively) and shown that they can physically interact and exchange electrons with both PFOR and OGOR, suggesting that they could be the physiological electron donors of the system in vivo. Shotgun proteomics indicated that all the enzymes assumed to be involved in the rTCA cycle are produced in the A. aeolicus cells. A number of additional enzymes, previously suggested to be part of a putative partial Wood-Ljungdahl pathway used for the synthesis of serine and glycine from CO2 were identified by mass spectrometry, but their abundance in the cell seems to be much lower than that of the rTCA cycle. Their possible involvement in carbon assimilation is discussed.

NMR-based metabolomic analysis of the physiological role of the electron-bifurcating FeFe-hydrogenase Hnd in Solidesulfovibrio fructosivorans under pyruvate fermentation.

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.

The electron-bifurcating FeFe-hydrogenase Hnd is involved in ethanol metabolism in Desulfovibrio fructosovorans grown on pyruvate.

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.

Electrochemical Characterization of a Complex FeFe Hydrogenase, the Electron-Bifurcating Hnd From Desulfovibrio fructosovorans.

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.

Hydrogenases and H2 metabolism in sulfate-reducing bacteria of the Desulfovibrio genus.

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.

A new mechanistic model for an O2-protected electron-bifurcating hydrogenase, Hnd from Desulfovibrio fructosovorans.

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 (Em = -450 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 µmol 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.

Phototrophic hydrogen production from a clostridial [FeFe] hydrogenase expressed in the heterocysts of the cyanobacterium Nostoc PCC 7120.

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.

Expression of terminal oxidases under nutrient-starved conditions in Shewanella oneidensis: detection of the A-type cytochrome c oxidase.

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.

A biochemical approach to study the role of the terminal oxidases in aerobic respiration in Shewanella oneidensis MR-1.

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.

H2-dependent azoreduction by Shewanella oneidensis MR-1: involvement of secreted flavins and both [Ni-Fe] and [Fe-Fe] hydrogenases.

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.

Drosophila translational elongation factor-1gamma is modified in response to DOA kinase activity and is essential for cellular viability.

Drosophila translational elongation factor-1gamma (EF1gamma) interacts in the yeast two-hybrid system with DOA, the LAMMER protein kinase of Drosophila. Analysis of mutant EF1gamma alleles reveals that the locus encodes a structurally conserved protein essential for both organismal and cellular survival. Although no genetic interactions were detected in combinations with mutations in EF1alpha, an EF1gamma allele enhanced mutant phenotypes of Doa alleles. A predicted LAMMER kinase phosphorylation site conserved near the C terminus of all EF1gamma orthologs is a phosphorylation site in vitro for both Drosophila DOA and tobacco PK12 LAMMER kinases. EF1gamma protein derived from Doa mutant flies migrates with altered mobility on SDS gels, consistent with it being an in vivo substrate of DOA kinase. However, the aberrant mobility appears to be due to a secondary protein modification, since the mobility of EF1gamma protein obtained from wild-type Drosophila is unaltered following treatment with several nonspecific phosphatases. Expression of a construct expressing a serine-to-alanine substitution in the LAMMER kinase phosphorylation site into the fly germline rescued null EF1gamma alleles but at reduced efficiency compared to a wild-type construct. Our data suggest that EF1gamma functions in vital cellular processes in addition to translational elongation and is a LAMMER kinase substrate in vivo.

Dissection of darkener of apricot kinase isoform functions in Drosophila.

The Darkener of apricot (Doa) locus of Drosophila encodes a LAMMER protein kinase affecting alterative splicing, and hence sex determination, via the phosphorylation of SR and SR-like proteins. Doa encodes 6 different kinases via alternative promoter usage. To provide further insight into the roles of the multiple isoforms, we mapped polymorphisms, deletions, and P-element insertions in the locus, identifying several that are largely, if not completely, isoform specific in their effects. These tests, along with the use of lines permitting overexpression and interfering RNA expression, demonstrate that the major isoforms of 55 and 105 kDa perform separate functions. The 105-kDa and a minor 138-kDa isoform are both vital but do not apparently perform functions essential for sex determination. Curiously, male-specific lethality induced by overexpression of the 55-kDa kinase in the larval fat body is rescued by coexpression of TRA, suggesting a sex-specific physiological role for this isoform. Maternal effects in which the survival of heteroallelic adults depends upon the direction of the cross are consistent with a role for a 105-kDa cytoplasmic kinase in oogenesis or early larval development.

Alternative promoter usage generates multiple evolutionarily conserved isoforms of Drosophila DOA kinase.

The unique LAMMER (or Clk) protein kinase of Drosophila is encoded at the Doa locus. To better understand the pleiotropic effects of Doa mutations, we describe the structure and expression of the multiple RNA and protein products of the locus, as well as their evolutionary conservation among Drosophila. The gene produces at least six different protein isoforms, primarily through alternative promoter usage, generating kinases with virtually identical catalytic domains but variable N-terminal noncatalytic domains. The single known alternative splicing event generates a kinase with the insertion of six additional amino-acids in the catalytic domain. Two independent predicted genes nested within Doa introns actually encode additional alternative N-termini of the locus. An alternative polyadenylation site utilized exclusively during early embryogenesis generates a transcript with a short half-life, apparently to ensure a "burst" of kinase expression at the onset of development. Ecdysone induction of Doa transcripts affects all isoforms during pupariation. Finally, extensive conservation of amino-acid sequences of both the catalytic and N-terminal noncatalytic exons observed in alignments between D. melanogaster exons and the genome sequences of 11 other Drosophila species suggest that the multiple isoforms serve important and nonredundant functions.

Polymorphisms in the C-terminal domain of MECP2 in mentally handicapped boys: implications for genetic counselling.

Numerous recent reports have proposed that mutations in the C-terminal domain of the MECP2 gene could be a frequent cause of mental retardation in males. We have identified two mutations in this particular domain (S359P and E397K) in two boys who were screened for MECP2 mutations in a series of 23 mentally handicapped boys fitting the clinical description of the previously reported cases. A detailed familial study based on three generations shows that the first mutation (S359P) was also inherited by a healthy cousin thus ruling out its involvement in the etiology of the phenotype of this patient. The second mutation (E397K) was also found in normal individuals. These findings clearly call for a careful consideration of the pathogenicity of the MECP2 mutations identified in sporadic male cases before genetic counselling or prenatal diagnosis is proposed to the corresponding families.