FocA and its central role in fine-tuning pH homeostasis of enterobacterial formate metabolism.

During enterobacterial mixed-acid fermentation, formate is generated from pyruvate by the glycyl-radical enzyme pyruvate formate-lyase (PflB). In Escherichia coli, especially at low pH, formate is then disproportionated to CO2 and H2 by the cytoplasmically oriented, membrane-associated formate hydrogenlyase (FHL) complex. If electron acceptors are available, however, formate is oxidized by periplasmically oriented, respiratory formate dehydrogenases. Formate translocation across the cytoplasmic membrane is controlled by the formate channel, FocA, a member of the formate-nitrite transporter (FNT) family of homopentameric anion channels. This review highlights recent advances in our understanding of how FocA helps to maintain intracellular formate and pH homeostasis during fermentation. Efflux and influx of formate/formic acid are distinct processes performed by FocA and both are controlled through protein interaction between FocA's N-terminal domain with PflB. Formic acid efflux by FocA helps to maintain cytoplasmic pH balance during exponential-phase growth. Uptake of formate against the electrochemical gradient (inside negative) is energetically and mechanistically challenging for a fermenting bacterium unless coupled with proton/cation symport. Translocation of formate/formic acid into the cytoplasm necessitates an active FHL complex, whose synthesis also depends on formate. Thus, FocA, FHL and PflB function together to govern formate homeostasis. We explain how FocA achieves efflux of formic acid and propose mechanisms for pH-dependent uptake of formate both with and without proton symport. We propose that FocA displays both channel- and transporter-like behaviour. Whether this translocation behaviour is shared by other members of the FNT family is also discussed.

A single amino acid exchange converts FocA into a unidirectional efflux channel for formate.

During mixed-acid fermentation, Escherichia coli initially translocates formate out of the cell, but re-imports it at lower pH. This is performed by FocA, the archetype of the formate-nitrite transporter (FNT) family of pentameric anion channels. Each protomer of FocA has a hydrophobic pore through which formate/formic acid is bidirectionally translocated. It is not understood how the direction of formate/formic acid passage through FocA is controlled by pH. A conserved histidine residue (H209) is located within the translocation pore, suggesting that protonation/deprotonation might be linked to the direction of formate translocation. Using a formate-responsive lacZ-based reporter system we monitored changes in formate levels in vivo when H209 in FocA was exchanged for either of the non-protonatable amino acids asparagine or glutamine, which occur naturally in some FNTs. These FocA variants (with N or Q) functioned as highly efficient formate efflux channels and the bacteria could neither accumulate formate nor produce hydrogen gas. Therefore, the data in this study suggest that this central histidine residue within the FocA pore is required for pH-dependent formate uptake into E. coli cells. We also address why H209 is evolutionarily conserved and provide a physiological rationale for the natural occurrence of N/Q variants of FNT channels.

Influence of C4 -Dcu transporters on hydrogenase and formate dehydrogenase activities in stationary phase-grown fermenting Escherichia coli.

During mixed-acid fermentation, Escherichia coli transports succinate mainly via transporters of the Dcu family. Here, we analyze the influence of Dcu transporters on hydrogenase (Hyd) and fermentative formate dehydrogenase (FDH-H) activities and how this is affected by external pH and carbon source. Using selected dcu mutations, it was shown that Dcu carriers mainly affect Hyd and FDH-H activities during glycerol but not glucose fermentation at acidic pH. During glycerol fermentation at pH 5.5, inactivation of either one or all Dcu carriers increased total Hyd activity by 60% compared with wild type. Under the same growth conditions, a dcuACBD mutant had a twofold higher FDH-H activity. When glucose was fermented in dcuD single mutant at pH 5.5, the FDH-H activity was also increased twofold compared with wild type. Interestingly, in dcuD or dcuACBD mutants at pH 7.5, Hyd activity was lowered by 20%. Taken together, it can be concluded that during glucose fermentation at pH 7.5, lack of DcuD affects Hyd enzyme activity, but at pH 5.5, it has a stronger effect on FDH-H activity. During glycerol fermentation, lack of Dcu carriers increased Hyd and FDH-H activities as revealed at pH 5.5. The results suggest that impairing Dcu transport function increases intracellular formate levels and thus affects H2 cycling and proton-motive force generation.

The N-terminal domains of the paralogous HycE and NuoCD govern assembly of the respective formate hydrogenlyase and NADH dehydrogenase complexes.

Formate hydrogenlyase (FHL) is the main hydrogen-producing enzyme complex in enterobacteria. It converts formate to CO2 and H2 via a formate dehydrogenase and a [NiFe]-hydrogenase. FHL and complex I are evolutionarily related and share a common core architecture. However, complex I catalyses the fundamentally different electron transfer from NADH to quinone and pumps protons. The catalytic FHL subunit, HycE, resembles NuoCD of Escherichia coli complex I; a fusion of NuoC and NuoD present in other organisms. The C-terminal domain of HycE harbours the [NiFe]-active site and is similar to other hydrogenases, while this domain in NuoCD is involved in quinone binding. The N-terminal domains of these proteins do not bind cofactors and are not involved in electron transfer. As these N-terminal domains are separate proteins in some organisms, we removed them in E. coli and observed that both FHL and complex I activities were essentially absent. This was due to either a disturbed assembly or to complex instability. Replacing the N-terminal domain of HycE with a 180 amino acid E. coli NuoC protein fusion did not restore activity, indicating that the domains have complex-specific functions. A FHL complex in which the N- and C-terminal domains of HycE were physically separated still retained most of its FHL activity, while the separation of NuoCD abolished complex I activity completely. Only the FHL complex tolerates physical separation of the HycE domains. Together, the findings strongly suggest that the N-terminal domains of these proteins are key determinants in complex assembly.

Delimiting the Function of the C-Terminal Extension of the Escherichia coli [NiFe]-Hydrogenase 2 Large Subunit Precursor.

The active site of all [NiFe]-hydrogenases (Hyd) has a bimetallic NiFe(CN)2CO cofactor that requires the combined action of several maturation proteins for its biosynthesis and insertion into the precursor form of the large subunit of the enzyme. Cofactor insertion is an intricately controlled process, and the large subunit of almost all Hyd enzymes has a C-terminal oligopeptide extension that is endoproteolytically removed as the final maturation step. This extension might serve either as one of the recognition motifs for the endoprotease, as well as an interaction platform for the maturation proteins, or it could have a structural role to ensure the active site cavity remains open until the cofactor is inserted. To distinguish between these alternatives, we exchanged the complete C-terminal extension of the precursor of Escherichia coli hydrogenase 2 (Hyd-2) for the C-terminal extension of the Hyd-1 enzyme. Using in-gel activity staining, we demonstrate clearly that this large subunit precursor retains its specificity for the HybG maturation chaperone, as well as for the pro-HybC-specific endoprotease HybD, despite the C-terminal exchange. Bacterial two-hybrid studies confirmed interaction between HybD and the pro-HybC variant carrying the exchanged C-terminus. Limited proteolysis studies of purified precursor and mature HybC protein revealed that, in contrast to the precursor, the mature protein was protected against trypsin attack, signifying a major conformational change in the protein. Together, our results support a model whereby the function of the C-terminal extension during subunit maturation is structural.

Amino acid variants of the HybB membrane subunit of Escherichia coli [NiFe]-hydrogenase-2 support a role in proton transfer.

[NiFe]-hydrogenase (Hyd) 2 of Escherichia coli has been proposed to generate proton motive force during H2 -oxidation, which it is dependent on if cells are incubated anaerobically with glycerol to drive reverse H2 -production. The integral membrane subunit HybB is required for proton transfer (PT) by Hyd-2 but has no cofactor. To provide evidence for PT by HybB, we analyzed the roles of conserved amino acid residues in a predicted proton channel. Exchange of conserved residues identified residues Y99, E133, H184, and E228 as mandatory for PT from the cytoplasm and quinol oxidation. In contrast, exchange of W54, D58, or R89 rendered Hyd-2 uni-directional and influenced the equilibrium. Our findings show that HybB is the key subunit in PT.

Bioenergetic aspects of archaeal and bacterial hydrogen metabolism.

Hydrogenases are metal-containing biocatalysts that reversibly convert protons and electrons to hydrogen gas. This reaction can contribute in different ways to the generation of the proton motive force (PMF) of a cell. One means of PMF generation involves reduction of protons on the inside of the cytoplasmic membrane, releasing H2 gas, which being without charge is freely diffusible across the cytoplasmic membrane, where it can be re-oxidized to release protons. A second route of PMF generation couples transfer of electrons derived from H2 oxidation to quinone reduction and concomitant proton uptake at the membrane-bound heme cofactor. This redox-loop mechanism, as originally formulated by Mitchell, requires a second, catalytically distinct, enzyme complex to re-oxidize quinol and release the protons outside the cell. A third way of generating PMF is also by electron transfer to quinones but on the outside of the membrane while directly drawing protons through the entire membrane. The cofactor-less membrane subunits involved are proposed to operate by a conformational mechanism (redox-linked proton pump). Finally, PMF can be generated through an electron bifurcation mechanism, whereby an exergonic reaction is tightly coupled with an endergonic reaction. In all cases the protons can be channelled back inside through a F1F0-ATPase to convert the 'energy' stored in the PMF into the universal cellular energy currency, ATP. New and exciting discoveries employing these mechanisms have recently been made on the bioenergetics of hydrogenases, which will be discussed here and placed in the context of their contribution to energy conservation.

Dissection of the Hydrogen Metabolism of the Enterobacterium Trabulsiella guamensis: Identification of a Formate-Dependent and Essential Formate Hydrogenlyase Complex Exhibiting Phylogenetic Similarity to Complex I.

Trabulsiella guamensis is a nonpathogenic enterobacterium that was isolated from a vacuum cleaner on the island of Guam. It has one H2-oxidizing Hyd-2-type hydrogenase (Hyd) and encodes an H2-evolving Hyd that is most similar to the uncharacterized Escherichia coli formate hydrogenlyase (FHL-2 Ec ) complex. The T. guamensis FHL-2 (FHL-2 Tg ) complex is predicted to have 5 membrane-integral and between 4 and 5 cytoplasmic subunits. We showed that the FHL-2 Tg complex catalyzes the disproportionation of formate to CO2 and H2 FHL-2 Tg has activity similar to that of the E. coli FHL-1 Ec complex in H2 evolution from formate, but the complex appears to be more labile upon cell lysis. Cloning of the entire 13-kbp FHL-2 Tg operon in the heterologous E. coli host has now enabled us to unambiguously prove FHL-2 Tg activity, and it allowed us to characterize the FHL-2 Tg complex biochemically. Although the formate dehydrogenase (FdhH) gene fdhF is not contained in the operon, the FdhH is part of the complex, and FHL-2 Tg activity was dependent on the presence of E. coli FdhH. Also, in contrast to E. coli, T. guamensis can ferment the alternative carbon source cellobiose, and we further investigated the participation of both the H2-oxidizing Hyd-2 Tg and the H2-forming FHL-2 Tg under these conditions.IMPORTANCE Biological H2 production presents an attractive alternative for fossil fuels. However, in order to compete with conventional H2 production methods, the process requires our understanding on a molecular level. FHL complexes are efficient H2 producers, and the prototype FHL-1 Ec complex in E. coli is well studied. This paper presents the first biochemical characterization of an FHL-2-type complex. The data presented here will enable us to solve the long-standing mystery of the FHL-2 Ec complex, allow a first biochemical characterization of T. guamensis's fermentative metabolism, and establish this enterobacterium as a model organism for FHL-dependent energy conservation.

Insights Into the Redox Sensitivity of Chloroflexi Hup-Hydrogenase Derived From Studies in Escherichia coli: Merits and Pitfalls of Heterologous [NiFe]-Hydrogenase Synthesis.

The highly oxygen-sensitive hydrogen uptake (Hup) hydrogenase from Dehalococcoides mccartyi forms part of a protein-based respiratory chain coupling hydrogen oxidation with organohalide reduction on the outside of the cell. The HupXSL proteins were previously shown to be synthesized and enzymatically active in Escherichia coli. Here we examined the growth conditions that deliver active Hup enzyme that couples H2 oxidation to benzyl viologen (BV) reduction, and identified host factors important for this process. In a genetic background lacking the three main hydrogenases of E. coli we could show that additional deletion of genes necessary for selenocysteine biosynthesis resulted in inactive Hup enzyme, suggesting requirement of a formate dehydrogenase for Hup activity. Hup activity proved to be dependent on the presence of formate dehydrogenase (Fdh-H), which is typically associated with the H2-evolving formate hydrogenlyase (FHL) complex in the cytoplasm. Further analyses revealed that heterologous Hup activity could be recovered if the genes encoding the ferredoxin-like electron-transfer protein HupX, as well as the related HycB small subunit of Fdh-H were also deleted. These findings indicated that the catalytic HupL and electron-transferring HupS subunits were sufficient for enzyme activity with BV. The presence of the HupX or HycB proteins in the absence of Fdh-H therefore appears to cause inactivation of the HupSL enzyme. This is possibly because HupX or HycB aided transfer of electrons to the quinone pool or other oxidoreductase complexes, thus maintaining the HupSL heterodimer in a continuously oxidized state causing its inactivation. This proposal was supported by the observation that growth under either aerobic or anaerobic respiratory conditions did not yield an active HupSL. These studies thus provide a system to understand the redox sensitivity of this heterologously synthesized hydrogenase.

The Extended C-Terminal α-Helix of the HypC Chaperone Restricts Recognition of Large Subunit Precursors by the Hyp-Scaffold Machinery during [NiFe]-Hydrogenase Maturation in Escherichia coli.

Members of the HypC protein family are chaperone-like proteins that play a central role in the maturation of [NiFe]-hydrogenases (Hyd). Escherichia coli has a second copy of HypC, called HybG, and, as a component of the HypDEF maturation scaffold, these proteins help synthesize the NiFe-cofactor and guide the scaffold to its designated hydrogenase large subunit precursor. HypC is required to synthesize active Hyd-1 and Hyd-3, while HybG facilitates Hyd-2 and Hyd-1 synthesis. To identify determinants on HypC that allow it to discriminate against Hyd-2, we made amino acid exchanges in 3 variable regions, termed VR1, VR2, and VR3, of HypC, that make it more similar to HybG. Region VR3 includes a HypC-specific C-terminal α-helical extension, and this proved particularly important in preventing the maturation of Hyd-2 by HypC. Truncation of this extension on HypC increased Hyd-2 activity in the absence of HybG, while retaining maturation of Hyd-3 and Hyd-1. Combining this truncation with amino acid exchanges in VR1 and VR2 of HypC negatively affected the synthesis of active Hyd-1. The C-terminus of E. coli HypC is thus a key determinant in hindering Hyd-2 maturation, while VR1 and VR2 appear more important for Hyd-1 matu-ration.

The Ferredoxin-Like Proteins HydN and YsaA Enhance Redox Dye-Linked Activity of the Formate Dehydrogenase H Component of the Formate Hydrogenlyase Complex.

Formate dehydrogenase H (FDH-H) and [NiFe]-hydrogenase 3 (Hyd-3) form the catalytic components of the hydrogen-producing formate hydrogenlyase (FHL) complex, which disproportionates formate to H2 and CO2 during mixed acid fermentation in enterobacteria. FHL comprises minimally seven proteins and little is understood about how this complex is assembled. Early studies identified a ferredoxin-like protein, HydN, as being involved in FDH-H assembly into the FHL complex. In order to understand how FDH-H and its small subunit HycB, which is also a ferredoxin-like protein, attach to the FHL complex, the possible roles of HydN and its paralogue, YsaA, in FHL complex stability and assembly were investigated. Deletion of the hycB gene reduced redox dye-mediated FDH-H activity to approximately 10%, abolished FHL-dependent H2-production, and reduced Hyd-3 activity. These data are consistent with HycB being an essential electron transfer component of the FHL complex. The FDH-H activity of the hydN and the ysaA deletion strains was reduced to 59 and 57% of the parental, while the double deletion reduced activity of FDH-H to 28% and the triple deletion with hycB to 1%. Remarkably, and in contrast to the hycB deletion, the absence of HydN and YsaA was without significant effect on FHL-dependent H2-production or total Hyd-3 activity; FDH-H protein levels were also unaltered. This is the first description of a phenotype for the E. coli ysaA deletion strain and identifies it as a novel factor required for optimal redox dye-linked FDH-H activity. A ysaA deletion strain could be complemented for FDH-H activity by hydN and ysaA, but the hydN deletion strain could not be complemented. Introduction of these plasmids did not affect H2 production. Bacterial two-hybrid interactions showed that YsaA, HydN, and HycB interact with each other and with the FDH-H protein. Further novel anaerobic cross-interactions of 10 ferredoxin-like proteins in E. coli were also discovered and described. Together, these data indicate that FDH-H activity measured with the redox dye benzyl viologen is the sum of the FDH-H protein interacting with three independent small subunits and suggest that FDH-H can associate with different redox-protein complexes in the anaerobic cell to supply electrons from formate oxidation.

The dual-function chaperone HycH improves assembly of the formate hydrogenlyase complex.

The assembly of multi-protein complexes requires the concerted synthesis and maturation of its components and subsequently their co-ordinated interaction. The membrane-bound formate hydrogenlyase (FHL) complex is the primary hydrogen-producing enzyme in Escherichia coli and is composed of seven subunits mostly encoded within the hycA-I operon for [NiFe]-hydrogenase-3 (Hyd-3). The HycH protein is predicted to have an accessory function and is not part of the final structural FHL complex. In this work, a mutant strain devoid of HycH was characterised and found to have significantly reduced FHL activity due to the instability of the electron transfer subunits. HycH was shown to interact specifically with the unprocessed species of HycE, the catalytic hydrogenase subunit of the FHL complex, at different stages during the maturation and assembly of the complex. Variants of HycH were generated with the aim of identifying interacting residues and those that influence activity. The R70/71/K72, the Y79, the E81 and the Y128 variant exchanges interrupt the interaction with HycE without influencing the FHL activity. In contrast, FHL activity, but not the interaction with HycE, was negatively influenced by H37 exchanges with polar residues. Finally, a HycH Y30 variant was unstable. Surprisingly, an overlapping function between HycH with its homologous counterpart HyfJ from the operon encoding [NiFe]-hydrogenase-4 (Hyd-4) was identified and this is the first example of sharing maturation machinery components between Hyd-3 and Hyd-4 complexes. The data presented here show that HycH has a novel dual role as an assembly chaperone for a cytoplasmic [NiFe]-hydrogenase.

Differential effects of isc operon mutations on the biosynthesis and activity of key anaerobic metalloenzymes in Escherichia coli.

Escherichia coli has two machineries for the synthesis of FeS clusters, namely Isc (iron-sulfur cluster) and Suf (sulfur formation). The Isc machinery, encoded by the iscRSUA-hscBA-fdx-iscXoperon, plays a crucial role in the biogenesis of FeS clusters for the oxidoreductases of aerobic metabolism. Less is known, however, about the role of ISC in the maturation of key multi-subunit metalloenzymes of anaerobic metabolism. Here, we determined the contribution of each iscoperon gene product towards the functionality of the major anaerobic oxidoreductases in E. coli, including three [NiFe]-hydrogenases (Hyd), two respiratory formate dehydrogenases (FDH) and nitrate reductase (NAR). Mutants lacking the cysteine desulfurase, IscS, lacked activity of all six enzymes, as well as the activity of fumaratereductase, and this was due to deficiencies in enzyme biosynthesis, maturation or FeS cluster insertion into electron-transfer components. Notably, based on anaerobic growth characteristics and metabolite patterns, the activity of the radical-S-adenosylmethionine enzyme pyruvate formate-lyase activase was independent of IscS, suggesting that FeS biogenesis for this ancient enzyme has different requirements. Mutants lacking either the scaffold protein IscU, the ferredoxin Fdx or the chaperones HscA or HscB had similar enzyme phenotypes: five of the oxidoreductases were essentially inactive, with the exception being the Hyd-3 enzyme, which formed part of the H2-producing formate hydrogenlyase (FHL) complex. Neither the frataxin-homologue CyaY nor the IscX protein was essential for synthesis of the three Hyd enzymes. Thus, while IscS is essential for H2 production in E. coli, the other ISC components are non-essential.

Expanding the substrates for a bacterial hydrogenlyase reaction.

Escherichia coli produces enzymes dedicated to hydrogen metabolism under anaerobic conditions. In particular, a formate hydrogenlyase (FHL) enzyme is responsible for the majority of hydrogen gas produced under fermentative conditions. FHL comprises a formate dehydrogenase (encoded by fdhF) linked directly to [NiFe]-hydrogenase-3 (Hyd-3), and formate is the only natural substrate known for proton reduction by this hydrogenase. In this work, the possibility of engineering an alternative electron donor for hydrogen production has been explored. Rational design and genetic engineering led to the construction of a fusion between Thermotoga maritima ferredoxin (Fd) and Hyd-3. The Fd-Hyd-3 fusion was found to evolve hydrogen when co-produced with T. maritima pyruvate :: ferredoxin oxidoreductase (PFOR), which links pyruvate oxidation to the reduction of ferredoxin. Analysis of the key organic acids produced during fermentation suggested that the PFOR/Fd-Hyd-3 fusion system successfully diverted pyruvate onto a new pathway towards hydrogen production.

Anaerobic Formate and Hydrogen Metabolism.

Numerous recent developments in the biochemistry, molecular biology, and physiology of formate and H2 metabolism and of the [NiFe]-hydrogenase (Hyd) cofactor biosynthetic machinery are highlighted. Formate export and import by the aquaporin-like pentameric formate channel FocA is governed by interaction with pyruvate formate-lyase, the enzyme that generates formate. Formate is disproportionated by the reversible formate hydrogenlyase (FHL) complex, which has been isolated, allowing biochemical dissection of evolutionary parallels with complex I of the respiratory chain. A recently identified sulfido-ligand attached to Mo in the active site of formate dehydrogenases led to the proposal of a modified catalytic mechanism. Structural analysis of the homologous, H2-oxidizing Hyd-1 and Hyd-5 identified a novel proximal [4Fe-3S] cluster in the small subunit involved in conferring oxygen tolerance to the enzymes. Synthesis of Salmonella Typhimurium Hyd-5 occurs aerobically, which is novel for an enterobacterial Hyd. The O2-sensitive Hyd-2 enzyme has been shown to be reversible: it presumably acts as a conformational proton pump in the H2-oxidizing mode and is capable of coupling reverse electron transport to drive H2 release. The structural characterization of all the Hyp maturation proteins has given new impulse to studies on the biosynthesis of the Fe(CN)2CO moiety of the [NiFe] cofactor. It is synthesized on a Hyp-scaffold complex, mainly comprising HypC and HypD, before insertion into the apo-large subunit. Finally, clear evidence now exists indicating that Escherichia coli can mature Hyd enzymes differentially, depending on metal ion availability and the prevailing metabolic state. Notably, Hyd-3 of the FHL complex takes precedence over the H2-oxidizing enzymes.

Exploring the directionality of Escherichia coli formate hydrogenlyase: a membrane-bound enzyme capable of fixing carbon dioxide to organic acid.

During mixed-acid fermentation Escherichia coli produces formate, which is initially excreted out the cell. Accumulation of formate, and dropping extracellular pH, leads to biosynthesis of the formate hydrogenlyase (FHL) complex. FHL consists of membrane and soluble domains anchored within the inner membrane. The soluble domain comprises a [NiFe] hydrogenase and a formate dehydrogenase that link formate oxidation directly to proton reduction with the release of CO2 and H2 . Thus, the function of FHL is to oxidize excess formate at low pH. FHL subunits share identity with subunits of the respiratory Complex I. In particular, the FHL membrane domain contains subunits (HycC and HycD) that are homologs of NuoL/M/N and NuoH, respectively, which have been implicated in proton translocation. In this work, strain engineering and new assays demonstrate unequivocally the nonphysiological reverse activity of FHL in vivo and in vitro. Harnessing FHL to reduce CO2 to formate is biotechnologically important. Moreover, assays for both possible FHL reactions provide opportunities to explore the bioenergetics using biochemical and genetic approaches. Comprehensive mutagenesis of hycC did not identify any single amino acid residues essential for FHL operation. However, the HycD E199, E201, and E203 residues were found to be critically important for FHL function.

SlyD-dependent nickel delivery limits maturation of [NiFe]-hydrogenases in late-stationary phase Escherichia coli cells.

Fermentatively growing Escherichia coli cells have three active [NiFe]-hydrogenases (Hyd), two of which, Hyd-1 and Hyd-2, contribute to H2 oxidation while Hyd-3 couples formate oxidation to H2 evolution. Biosynthesis of all Hyd involves the insertion of a Fe(CN)2CO group and a subsequent insertion of nickel ions through the HypA/HybF, HypB and SlyD proteins. With high nickel concentrations the presence of none of these proteins is required, but under normal growth conditions and during late stationary growth SlyD is important for hydrogenase activities. The slyD mutation reduced H2 production during exponential phase growth by about 50%. Assaying stationary phase grown cells for the coupling of Hyd activity to the respiratory chain or formate-dependent H2 evolution showed that SlyD is essential for both H2 evolution and H2 oxidation. Although introduction of plasmid-coded slyD resulted in an overall decrease of Hyd-2 polypeptides in slyD and hypA slyD mutants, processing and dye-reducing activity of the Hyd-2 enzyme was nevertheless restored. Similarly, introduction of the slyD plasmid restored only some H2 evolution in the slyD mutant while Hyd-3 polypeptides and dye-reducing activity were fully restored. Taken together, these results indicate an essential role for SlyD in the generation of the fully cofactor-equipped hydrogenase large subunits in the stationary phase where the level of each Hyd enzyme is finely tuned by SlyD for optimal enzyme activity.

Physiology and bioenergetics of [NiFe]-hydrogenase 2-catalyzed H2-consuming and H2-producing reactions in Escherichia coli.

Escherichia coli uptake hydrogenase 2 (Hyd-2) catalyzes the reversible oxidation of H2 to protons and electrons. Hyd-2 synthesis is strongly upregulated during growth on glycerol or on glycerol-fumarate. Membrane-associated Hyd-2 is an unusual heterotetrameric [NiFe]-hydrogenase that lacks a typical cytochrome b membrane anchor subunit, which transfers electrons to the quinone pool. Instead, Hyd-2 has an additional electron transfer subunit, termed HybA, with four predicted iron-sulfur clusters. Here, we examined the physiological role of the HybA subunit. During respiratory growth with glycerol and fumarate, Hyd-2 used menaquinone/demethylmenaquinone (MQ/DMQ) to couple hydrogen oxidation to fumarate reduction. HybA was essential for electron transfer from Hyd-2 to MQ/DMQ. H2 evolution catalyzed by Hyd-2 during fermentation of glycerol in the presence of Casamino Acids or in a fumarate reductase-negative strain growing with glycerol-fumarate was also shown to be dependent on both HybA and MQ/DMQ. The uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) inhibited Hyd-2-dependent H2 evolution from glycerol, indicating the requirement for a proton gradient. In contrast, CCCP failed to inhibit H2-coupled fumarate reduction. Although a Hyd-2 enzyme lacking HybA could not catalyze Hyd-2-dependent H2 oxidation or H2 evolution in whole cells, reversible H2-dependent reduction of viologen dyes still occurred. Finally, hydrogen-dependent dye reduction by Hyd-2 was reversibly inhibited in extracts derived from cells grown in H2 evolution mode. Our findings suggest that Hyd-2 switches between H2-consuming and H2-producing modes in response to the redox status of the quinone pool. Hyd-2-dependent H2 evolution from glycerol requires reverse electron transport.

Integration of an [FeFe]-hydrogenase into the anaerobic metabolism of Escherichia coli.

Biohydrogen is a potentially useful product of microbial energy metabolism. One approach to engineering biohydrogen production in bacteria is the production of non-native hydrogenase activity in a host cell, for example Escherichia coli. In some microbes, hydrogenase enzymes are linked directly to central metabolism via diaphorase enzymes that utilise NAD+/NADH cofactors. In this work, it was hypothesised that heterologous production of an NAD+/NADH-linked hydrogenase could connect hydrogen production in an E. coli host directly to its central metabolism. To test this, a synthetic operon was designed and characterised encoding an apparently NADH-dependent, hydrogen-evolving [FeFe]-hydrogenase from Caldanaerobacter subterranus. The synthetic operon was stably integrated into the E. coli chromosome and shown to produce an active hydrogenase, however no H2 production was observed. Subsequently, it was found that heterologous co-production of a pyruvate::ferredoxin oxidoreductase and ferredoxin from Thermotoga maritima was found to be essential to drive H2 production by this system. This work provides genetic evidence that the Ca.subterranus [FeFe]-hydrogenase could be operating in vivo as an electron-confurcating enzyme.