The roles of hydrogenases 3 and 4, and the F0F1-ATPase, in H2 production by Escherichia coli at alkaline and acidic pH

The hyc operon of Escherichia coli encodes the H2-evolving hydrogenase 3 (Hyd-3) complex that, in conjunction with formate dehydrogenase H (Fdh-H), constitutes a membrane-associated formate hydrogenlyase (FHL) catalyzing the disproportionation of formate to CO2 and H2 during fermentative growth at low pH. Recently, an operon (hyf) encoding a potential second H2-evolving hydrogenase (Hyd-4) was identified in E. coli. In this study the roles of the hyc- and hyf-encoded systems in formate-dependent H2 production and Fdh-H activity have been investigated. In cells grown on glucose under fermentative conditions at slightly acidic pH the production of H2 was mostly Hyd-3- and Fdh-H-dependent, and Fdh-H activity was also mainly Hyd-3-dependent. However, at slightly alkaline pH, H2 production was found to be largely Hyd-4, Fdh-H and F0F1-ATPase-dependent, and Fdh-H activity was partially dependent on Hyd-4 and F0F1-ATPase. These results suggest that, at slightly alkaline pH, H2 production and Fdh-H activity are dependent on both the F0F1-ATPase and a novel FHL, designated FHL-2, which is composed of Hyd-4 and Fdh-H, and is driven by a proton gradient established by the F0F1-ATPase.     

Hydrogenase 3 but not hydrogenase 4 is major in hydrogen gas production by Escherichia coli formate hydrogenlyase at acidic pH and in the presence of external formate

Fermenting Escherichia coli is able to produce formate and molecular hydrogen (H₂) when grown on glucose. H₂ formation is possessed by two hydrogenases, 3 (Hyd-3) and 4 (Hyd-4), those, in conjunction with formate dehydrogenase H (Fdh-H), constitute distinct membrane-associated formate hydrogenylases. At slightly alkaline pH (pH 7.5), the production of H₂ was found to be dependent on Hyd-4 and the F₀F₁-adenosine triphosphate (ATPase), whereas external formate increased the activity of Hyd-3. In this study with cells grown without and with external formate H₂ production dependent on pH was investigated. In both types of cells, H₂ production was increased after lowering of pH. At acidic pH (pH 5.5), this production became insensitive either to N,N′-dicyclohexylcarbodiimide or to osmotic shock and it became largely dependent on Fdh-H and Hyd-3 but not Hyd-4 and the F₀F₁-ATPase. The results indicate that Hyd-3 has a major role in H₂ production at acidic pH independently on the F₀F₁-ATPase.     

Hydrogenase 3 but not hydrogenase 4 is major in hydrogen gas production by Escherichia coli formate hydrogenlyase at acidic pH and in the presence of external formate

Fermenting Escherichia coli is able to produce formate and molecular hydrogen (H2) when grown on glucose. H2 formation is possessed by two hydrogenases, 3 (Hyd-3) and 4 (Hyd-4), those, in conjunction with formate dehydrogenase H (Fdh-H), constitute distinct membrane-associated formate hydrogenylases. At slightly alkaline pH (pH 7.5), the production of H2 was found to be dependent on Hyd-4 and the F0F1-adenosine triphosphate (ATPase), whereas external formate increased the activity of Hyd-3. In this study with cells grown without and with external formate, H2 production dependent on pH was investigated. In both types of cells, H2 production was increased after lowering of pH. At acidic pH (pH 5.5), this production became insensitive either to N,N'-dicyclohexylcarbodiimide or to osmotic shock and it became largely dependent on Fdh-H and Hyd-3 but not Hyd-4 and the F0F1-ATPase. The results indicate that Hyd-3 has a major role in H2 production at acidic pH independently on the F0F1-ATPase.     

Multiple and reversible hydrogenases for hydrogen production by Escherichia coli: dependence on fermentation substrate,pH and the F0F1-ATPase

Molecular hydrogen (H₂) can be produced via hydrogenases during mixed-acid fermentation by bacteria. Escherichia coli possesses multiple (four) hydrogenases. Hydrogenase 3 (Hyd-3) and probably 4 (Hyd-4) with formate dehydrogenase H (Fdh-H) form two different H₂-evolving formate hydrogen lyase (FHL) pathways during glucose fermentation. For both FHL forms, the hycB gene coding small subunit of Hyd-3 is required. Formation and activity of FHL also depends on the external pH ([pH]_out) and the presence of formate. FHL is related with the F₀F₁-ATPase by supplying reducing equivalents and depending on proton-motive force. Two other hydrogenases, 1 (Hyd-1) and 2 (Hyd-2), are H₂-oxidizing enzymes during glucose fermentation at neutral and low [pH]_out. They operate in a reverse, H₂-producing mode during glycerol fermentation at neutral [pH]_out. Hyd-1 and Hyd-2 activity depends on F₀F₁. Moreover, Hyd-3 can also work in a reverse mode. Therefore, the operation direction and activity of all Hyd enzymes might determine H₂ production; some metabolic cross-talk between Hyd enzymes is proposed. Manipulating of different Hyd enzymes activity is an effective way to enhance H₂ production by bacteria in biotechnology. Moreover, a novel approach would be the use of glycerol as feedstock in fermentation processes leading to H₂ production, reduced fuels and other chemicals with higher yields than those obtained by common sugars.     

Dependence on the F<Subscript>0</Subscript>F<Subscript>1</Subscript>-ATP synthase for the activities of the hydrogen-oxidizing hydrogenases 1 and 2 during glucose and glycerol fermentation at high and low pH in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Escherichia coli has four [NiFe]-hydrogenases (Hyd); three of these, Hyd-1, Hyd-2 and Hyd-3 have been characterized well. In this study the requirement for the F₀F₁-ATP synthase for the activities of the hydrogen-oxidizing hydrogenases Hyd-1 and Hyd-2 was examined. During fermentative growth on glucose at pH 7.5 an E. coli F₀F₁-ATP synthase mutant (DK8) lacked hydrogenase activity. At pH 5.5 hydrogenase activity was only 20% that of the wild type. Using in-gel activity staining, it could be demonstrated that both Hyd-1 and Hyd-2 were essentially inactive at these pHs, indicating that the residual activity at pH 5.5 was due to the hydrogen-evolving Hyd-3 enzyme. During fermentative growth in the presence of glycerol, hydrogenase activity in the mutant was highest at pH 7.5 attaining a value of 0.76 U/mg, or ~50% of wild type activity, and Hyd-2 was only partially active at this pH, while Hyd-1 was inactive. Essentially no hydrogenase activity was measured at pH 5.5 during growth with glycerol. At this pH the mutant had a hydrogenase activity that was maximally only ~10% of wild type activity with either carbon substrate but a weak activity of both Hyd-1 and Hyd-2 could be detected. Taken together, these results demonstrate for the first time that the activity of the hydrogen-oxidizing hydrogenases in E. coli depends on an active F₀F₁-ATP synthase during growth at high and low pH.     

Multiple and reversible hydrogenases for hydrogen production by Escherichia coli: dependence on fermentation substrate, pH and the F(0)F(1)-ATPase

Molecular hydrogen (H(2)) can be produced via hydrogenases during mixed-acid fermentation by bacteria. Escherichia coli possesses multiple (four) hydrogenases. Hydrogenase 3 (Hyd-3) and probably 4 (Hyd-4) with formate dehydrogenase H (Fdh-H) form two different H(2)-evolving formate hydrogen lyase (FHL) pathways during glucose fermentation. For both FHL forms, the hycB gene coding small subunit of Hyd-3 is required. Formation and activity of FHL also depends on the external pH ([pH](out)) and the presence of formate. FHL is related with the F(0)F(1)-ATPase by supplying reducing equivalents and depending on proton-motive force. Two other hydrogenases, 1 (Hyd-1) and 2 (Hyd-2), are H(2)-oxidizing enzymes during glucose fermentation at neutral and low [pH](out). They operate in a reverse, H(2)-producing mode during glycerol fermentation at neutral [pH](out). Hyd-1 and Hyd-2 activity depends on F(0)F(1). Moreover, Hyd-3 can also work in a reverse mode. Therefore, the operation direction and activity of all Hyd enzymes might determine H(2) production; some metabolic cross-talk between Hyd enzymes is proposed. Manipulating of different Hyd enzymes activity is an effective way to enhance H(2) production by bacteria in biotechnology. Moreover, a novel approach would be the use of glycerol as feedstock in fermentation processes leading to H(2) production, reduced fuels and other chemicals with higher yields than those obtained by common sugars.     

Chromogenic assessment of the three molybdo-selenoprotein formate dehydrogenases in Escherichia coli

Escherichia coli synthesizes three selenocysteine-dependent formate dehydrogenases (Fdh) that also have a molybdenum cofactor. Fdh-H couples formate oxidation with proton reduction in the formate hydrogenlyase (FHL) complex. The activity of Fdh-H in solution can be measured with artificial redox dyes but, unlike Fdh-O and Fdh-N, it has never been observed by chromogenic activity staining after non-denaturing polyacrylamide gel electrophoresis (PAGE). Here, we demonstrate that Fdh-H activity is present in extracts of cells from stationary phase cultures and forms a single, fast-migrating species. The activity is oxygen labile during electrophoresis explaining why it has not been previously observed as a discreet activity band. The appearance of Fdh-H activity was dependent on an active selenocysteine incorporation system, but was independent of the [NiFe]-hydrogenases (Hyd), 1, 2 or 3. We also identified new active complexes of Fdh-N and Fdh-O during fermentative growth. The findings of this study indicate that Fdh-H does not form a strong complex with other Fdh or Hyd enzymes, which is in line with it being able to deliver electrons to more than one redox-active enzyme complex.     

Hydrogenase-3 Contributes to Anaerobic Acid Resistance of Escherichia coli

Background

Hydrogen production by fermenting bacteria such as Escherichia coli offers a potential source of hydrogen biofuel. Because H₂ production involves consumption of 2H⁺, hydrogenase expression is likely to involve pH response and regulation. Hydrogenase consumption of protons in E. coli has been implicated in acid resistance, the ability to survive exposure to acid levels (pH 2–2.5) that are three pH units lower than the pH limit of growth (pH 5–6). Enhanced survival in acid enables a larger infective inoculum to pass through the stomach and colonize the intestine. Most acid resistance mechanisms have been defined using aerobic cultures, but the use of anaerobic cultures will reveal novel acid resistance mechanisms.

Methods and Principal Findings

We analyzed the pH regulation of bacterial hydrogenases in live cultures of E. coli K-12 W3110. During anaerobic growth in the range of pH 5 to 6.5, E. coli expresses three hydrogenase isoenzymes that reversibly oxidize H₂ to 2H⁺. Anoxic conditions were used to determine which of the hydrogenase complexes contribute to acid resistance, measured as the survival of cultures grown at pH 5.5 without aeration and exposed for 2 hours at pH 2 or at pH 2.5. Survival of all strains in extreme acid was significantly lower in low oxygen than for aerated cultures. Deletion of hyc (Hyd-3) decreased anoxic acid survival 3-fold at pH 2.5, and 20-fold at pH 2, but had no effect on acid survival with aeration. Deletion of hyb (Hyd-2) did not significantly affect acid survival. The pH-dependence of H₂ production and consumption was tested using a H₂-specific Clark-type electrode. Hyd-3-dependent H₂ production was increased 70-fold from pH 6.5 to 5.5, whereas Hyd-2-dependent H₂ consumption was maximal at alkaline pH. H₂ production, was unaffected by a shift in external or internal pH. H₂ production was associated with hycE expression levels as a function of external pH.

Conclusions

Anaerobic growing cultures of E. coli generate H₂ via Hyd-3 at low external pH, and consume H₂ via Hyd-2 at high external pH. Hyd-3 proton conversion to H₂ is required for acid resistance in anaerobic cultures of E. coli.     

Formate hydrogenlyase,formic acid translocation and hydrogen production: dynamic membrane biology during fermentation

Formate hydrogenlyase-1 (FHL-1) is a complex-I-like enzyme that is commonly found in gram-negative bacteria. The enzyme comprises a peripheral arm and a membrane arm but is not involved in quinone reduction. Instead, FHL-1 couples formate oxidation to the reduction of protons to molecular hydrogen (H₂). Escherichia coli produces FHL-1 under fermentative conditions where it serves to detoxify formic acid in the environment. The membrane biology and bioenergetics surrounding E. coli FHL-1 have long held fascination. Here, we review recent work on understanding the molecular basis of formic acid efflux and influx. We also consider the structure and function of E. coli FHL-1, its relationship with formate transport, and pay particular attention to the molecular interface between the peripheral arm and the membrane arm. Finally, we highlight the interesting phenotype of genetic mutation of the ND1 Loop, which is located at that interface.     

Carbon monoxide conversion to formate byMethanosarcina barkeri

Summary A preliminary study of formate production from CO plus H₂O using the intact cells ofMethanosarcina barkeri was conducted. Formate production from CO gas required the participation of three enzymes, CO dehydrogenase, hydrogenase and formate dehydrogenase. Hypophosphite inhibited formate formation from CO plus H₂O by about 80%. In this system, 9 g/l of formate could be obtained from CO gas after 48 h of incubation at 37°C, pH 8.0.     

Oxygen consumption by Campylobacter sputorum subspecies Bubulus with formate as substrate

The kinetics of oxygen utilization by the microaerophile Campylobacter sputorum subspecies bubulus was studied. With formate as substrate two enzyme systems were found to be responsible for electron transfer between formate and oxygen. In the case of lactate oxidation one enzyme system could account for the activity measured. One of the formateoxidizing systems possessed a high affinity for oxygen [K _m(O₂)=approx. 4M O₂]. From inhibitor studies it was concluded that a respiratory chain was involved in its activity. Respiration by this system must be responsible for proton translocation and electron transport-linked phosphorylation at formate oxidation. The other enzyme system had an extremely low affinity for oxygen [K _m (O₂)=approx. 1 mM O₂]. It was tentatively identified as the H₂O₂-producing formate oxidase previously found in C. sputorum. The H₂O₂ production by this enzyme is implicated in an explanation of the microaerophilic nature of C. sputorum. Sensitivity of formate dehydrogenase to H₂O₂ was demonstrated. The influence of the formate concentration on aerobic formate oxidation was determined. The pH- and temperature dependencies of oxygen uptake with formate as substrate were examined at airsaturation and at a low dissolved oxygen tension.Abbreviations TL medium tryptose-lactate medium - TF medium tryptose-formate medium - HQNO 2-n-heptyl-4-hydroxyquinoline N-oxide - SHAM salicylhydroxamic acid - DCPIP 2,6-dichlorophenolindophenol     

Characterization of <Emphasis Type="Italic">Escherichia coli</Emphasis> [NiFe]-Hydrogenase Distribution During Fermentative Growth at Different pHs

The contribution made by each of the three active [NiFe]-hydrogenases (Hyd) of Escherichia coli during fermentation of glucose or glycerol in peptone-based medium at different pHs was analysed. The activities of the hydrogen-oxidizing Hyd-1 and Hyd-2 enzymes showed a reciprocal dependence on the pH of the medium while Hyd-3, a key component of the hydrogen-evolving formate hydrogenlyase complex, was mainly active at pH 6.5. Our findings identify the conditions during fermentation of glucose or glycerol under which each [NiFe]-hydrogenase is optimally active and demonstrate a previously unrecognized dependence on Hyd-1 activity at low pH.     

Strain DCB-1 conserves energy for growth from reductive dechlorination coupled to formate oxidation

Strain DCB-1 is a strict anaerobe capable of the reductive dechlorination of chlorobenzoates. The effect of dechlorination on the yield of pure cultures of DCB-1 was tested. Cultures were incubated with formate or H₂ as electron donors and CO₂ as a putative carbon source. Relative to control cultures with benzoate, cultures which dechlorinated 3-chlorobenzoate and 3,5-dichlorobenzoate had higher yields measured both as protein and cell density. On the media tested the apparent growth yield was 1.7 to 3.4 g cell protein per mole Cl^- removed. Dechlorination also stimulated formate oxidation by growing cultures. Resuspended cells required an electron donor for dechlorination activity, with either formate or elemental iron serving this function. Resuspended cells did not require an electron acceptor for formate consumption, but reductive dechlorination of 3CB to benzoate stoichiometrically stimulated oxidation of formate to CO₂. These results indicate that DCB-1 conserves energy for growth by coupling formate, and probably, H₂ oxidation to reductive dechlorination.Non-standard abbreviations 3CB 3-chlorobenzoate - 35DCB 3,5-dichlorobenzoate - PCF Propionibacterium sp. culture fluid     

Contribution of Hydrogenase 2 to Stationary Phase H2 Production by Escherichia coli During Fermentation of Glycerol

Escherichia coli has four hydrogenases (Hyd), three genes of which are encoded by the hya, hyb, and hyc operons. The proton-reducing and hydrogen-oxidizing activities of Hyd-2 (hyb) were analyzed in whole cells grown to stationary phase and cell extracts, respectively, during glycerol fermentation using novel double mutants. H₂ production rate at pH 7.5 was decreased by ~3.5- and ~7-fold in hya and hyc (HDK 103) or hyb and hyc (HDK 203) operon double mutants, respectively, compared with the wild type. At pH 6.5, H₂ production decreased by ~2- and ~5-fold in HDK103 and HDK203, respectively, compared with the wild type. At pH 5.5, H₂ production was reduced by ~4.5-fold in the mutants compared with the wild type. The total hydrogen-oxidizing activity was shown to depend on the pH of the growth medium in agreement with previous findings and was significantly reduced in the HDK103 or HDK203 mutants. At pH 7.5, Hyd-2 activity was 0.26 U (mg protein)^?1 and Hyd-1 activity was 0.1 U (mg protein)^?1. As the pH of the growth medium decreased to 6.5, Hyd-2 activity was 0.16 U (mg protein)^?1, and Hyd-1 was absent. Surprisingly, at pH 5.5, there was an increase in Hyd-2 activity (0.33 U mg protein)^?1 but not in that of Hyd-1. These findings show a major contribution of Hyd-2 to H₂ production during glycerol fermentation that resulted from altered metabolism which surprisingly influenced proton reduction.     

Energetics and regulations of formate and hydrogen metabolism by Methanobacterium formicicum

Accumulation of formate to millimolar levels was observed during the growth of Methanobacterium formicicum species on H₂–CO₂. Hydrogen was also produced during formate metabolism by M. formicicum. The amount of formate accumulated in the medium or the amount H₂ released in gas phase was influenced by the bicarbonate concentration. The formate hydrogenlyase system was constitutive but regulated by formate. When methanogenesis was inhibited by addition of 2-bromoethane sulfonate, M. formicicum synthesized formate from H₂ plus HCO _inf3 ^sup- or produced H₂ from formate to a steady-state level at which point the Gibbs free energy (G) available for formate synthesis or H₂ production was approximately -2 to -3 kJ/reaction. Formate conversion to methane was inhibited in the presence of high H₂ pressure. The relative rates of conversion of formate and H₂ were apparently controlled by the G available for formate synthesis, hydrogen production, methane production from formate and methane production from H₂. Results from ¹⁴C-tracer tests indicated that a rapid isotopic exchange between HCOO^- and HCO _inf3 ^sup- occurred during the growth of M. formicicum on H₂–CO₂. Data from metabolism of ¹⁴C-labelled formate to methane suggested that formate was initially split to H₂ and HCO _inf3 ^sup- and then subsequently converted to methane. When molybdate was replaced with tungstate in the growth media, the growth of M. formicicum strain MF on H₂–CO₂ was inhibited although production of methane was not Formate synthesis from H₂ was also inhibited.     

Nicotinamide adenine dinucleotide-dependent formate dehydrogenase from Rhodopseudomonas palustris

Summary Formate dehydrogenase in extracts of the facultative phototroph, Rhodopseudomonas palustris was shown to be soluble and NAD-linked. The flavin nucleotides, FMN and FAD, stimulated the rate of NAD reduction about fourfold. Reduction of artificial electron acceptors such as DCPIP and cytochrome c was also stimulated by FMN and FAD. The pH optimum for the reduction of NAD was pH 8.0, in contrast to pH 6.8 for cytochrome c and DCPIP reduction. The apparent K _m for formate as measured by NAD reduction was 2.6×10^-4 M. Although the addition of thiosulfate or yeast extract to the formate medium increased both the growth rate and yield of Rhps. palustris, they had little effect on the activity of formate dehydrogenase.     

Hydrogenases and formate dehydrogenases of Syntrophobacter fumaroxidans

The syntrophic propionate-oxidizing bacterium Syntrophobacter fumaroxidans possesses two distinct formate dehydrogenases and at least three distinct hydrogenases. All of these reductases are either loosely membrane-associated or soluble proteins and at least one of the hydrogenases is located in the periplasm. These enzymes were expressed on all growth substrates tested, though the levels of each enzyme showed large variations. These findings suggest that both H₂ and formate are involved in the central metabolism of the organism, and that both these compounds may serve as interspecies electron carriers during syntrophic growth on propionate. This revised version was published online in August 2006 with corrections to the Cover Date.     

Energy transformation coupled to formate oxidation during anaerobic fermentation

A correlation between the rate of ATP synthesis by F₀F₁ ATP synthase and formate oxidation by formate hydrogen lyase (FHL) has been found in inside-out membrane vesicles of the Escherichia coli mutant JW 136 (Δhya/Δhyb) with double deletions of hydrogenases 1 and 2, grown anaerobically on glucose in the absence of external electron acceptors at pH 6.5. ATP synthesis was suppressed by the H⁺-ATPase inhibitors N,N′-dicyclohexylcarbodiimide, sodium azide, and the uncoupler carbonyl cyanide m-chlorophenylhydrazone. Copper ions inhibited formate-dependent hydrogenase and ATP-synthase activities but did not affect the ATPase activity of the vesicles. The maximal rate of ATP synthesis (0.83 μmol/min per mg protein) was determined at simultaneous application of sodium formate, ADP, and inorganic phosphate, and was stimulated by K⁺ ions. The results confirm the assumption of a dual role of hydrogenase 3, the formate hydrogen lyase subunit that can couple the reduction of protons to H₂ and their translocation through membrane with chemiosmotic synthesis of ATP.