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Anaerobic Formate and Hydrogen Metabolism

R. GARY SAWERS,¹ MELANIE BLOKESCH,² AND AUGUST BÖCK²*

[SECTION EDITOR, GEORGES N. COHEN]

Posted September 9, 2004

¹Department of Molecular Microbiology, John Innes Centre, Norwich, United Kingdom, and ²Department of Biology I—Microbiology, University of Munich, Maria Ward Strasse 1a, 80638 Munich, Germany

*Corresponding author: Phone: 49-89-21806116, E-mail: This e-mail address is being protected from spambots. You need JavaScript enabled to view it

 

INTRODUCTION

During fermentative growth on carbohydrates, Escherichia coli degrades the substrate via the glycolytic route into two pyruvate molecules. Pyruvate can be reduced to lactate or nonoxidatively cleaved by pyruvate formate lyase into acetyl-coenzyme A (acetyl-CoA) and formate. Acetyl-CoA can be utilized for energy conservation in the phosphotransacetylase (PTA) and acetate kinase (ACK) reaction sequence or can serve as an acceptor for reducing equivalents gathered during pyruvate formation, through the action of alcohol dehydrogenase (AdhE) ( Chapter Fermentative Pyruvate and Acetyl-Coenzyme A Metabolism). These branches of the fermentation pathway are present in many microorganisms that are able to grow under anaerobic conditions. In contrast, the conversion of one third of the carbon from glucose into the strongly acidic formic acid is characteristic of E. coli and its relatives and has attracted the attention of microbial physiologists for decades.

Formic acid has a redox potential of –420 mV under standard conditions and therefore can be classified as a high-energy compound. Its disproportionation into CO₂ and molecular hydrogen (E_m,7 –420 mV) via the formate hydrogenlyase (FHL) system is therefore of high selective value. The seemingly simple FHL reaction, however, is very complex and involves the participation of at least seven proteins, most of which are metalloenzymes, with requirements for iron, molybdenum, nickel, or selenium. Complex auxiliary systems are involved in the incorporation of these metals. Reutilization of the hydrogen evolved required the evolution of H₂ oxidation systems, which couple the oxidation process to an appropriate energy-conserving terminal reductase. E. coli has two such hydrogen reutilization systems. Finally, fermentation is the "last resort" of energy metabolism, since it gives the minimal energy yield when compared with respiratory processes. Consequently, fermentation is only used when external electron acceptors are absent. This has necessitated the establishment of regulatory cascades, which ensure that the metabolic capability is appropriately adjusted to the physiological condition. In this chapter we review the genetics, biochemistry, and regulation of the FHL system, of the H₂-oxidizing hydrogenases, and of the hydrogenase maturation system. A separate  (Chapter Selenocysteine) deals with the incorporation of selenium into proteins.

 

BIOCHEMISTRY OF COMPONENTS INVOLVED IN FORMATE AND HYDROGEN METABOLISM

 

Hydrogenases 1 and 2

It is not possible to distinguish hydrogenase isoenzyme 1 (Hyd-1), Hyd-2, or Hyd-3 on the basis of enzyme activity determination because the assay for all three measures the dihydrogen-dependent reduction of benzyl viologen (BV) (11, 34, 51, 67, 101). The isoenzymes are, however, distinguishable immunologically (15, 123). Both Hyd-1 and Hyd-2 (EC 1.12.1.-) have been purified (2, 16, 36, 42, 124) and their properties are summarized in Table 1. Hyd-2 can be purified as a soluble, active, tryptic fragment that differs from the native membrane-bound enzyme only through the loss of a 5-kDa fragment from the small subunit (16). The data indicate that the enzyme is anchored in the membrane, but a large proportion of the enzyme is exposed at the periplasmic surface of the membrane (108, 118). In contrast, Hyd-1 cannot be released from the membrane fraction by proteolysis, but it can be readily solubilized through the action of detergents such as Triton X-100 (Fig. 1) (15, 36, 123, 124). The large subunits of both Hyd-1 and Hyd-2 are subject to processing (see below) (16, 124), which has been shown to occur C-terminally and is part of the maturation process (27). Spectroscopic analysis of Hyd-1 confirmed that it has electron paramagnetic resonance (EPR) features typical of Ni-Fe hydrogenases (36).

Neither purified Hyd-1 nor Hyd-2 reacts with quinones, although the quinone pool might be expected to receive the electrons derived from dihydrogen oxidation catalyzed by these enzymes (see below). This suggests that a further subunit involved in electron transfer to the quinone pool may have been lost during purification (38, 121). Both Hyd-1 and Hyd-2 have a very low apparent K_m for dihydrogen and this is in accord with the enzymes' function in H₂ oxidation (16, 124). Hyd-2 has a greater capacity for dihydrogen oxidation than Hyd-1 (16).

 

The Formate Hydrogenlyase System

The FHL pathway was first described by Stephenson and Stickland (137, 138), and as the name suggests, it catalyzes the disproportionation of formate to carbon dioxide and dihydrogen. Studies in the 1950s identified an absolute requirement of selenium and molybdenum for the synthesis of active formate dehydrogenase (FDH) and hydrogen gas production by E. coli (103) and it was established that a FDH, a hydrogenase (Hyd), and two electron carriers constituted the FHL pathway (50, 101). The FDH isoenzyme associated with the FHL pathway is termed FDH-H and the Hyd isoenzyme is called Hyd-3. Both of these enzymes are biochemically and genetically distinct from the other FDH and Hyd isoenzymes present in E. coli and Salmonella enterica serovar Typhimurium (reviewed in reference 121). Current evidence indicates that the FHL pathway constitutes a multiprotein complex located on the inner aspect of the cytoplasmic membrane and henceforth will be referred to as the FHL complex (Fig. 1) (120).

 

Biochemistry of FDH-H

FHL complex activity can be determined as the formate-dependent production of dihydrogen (123), or the activity of the FDH-H isoenzyme (EC 1.2.1.-) component can be determined in isolation by measuring the formate-dependent reduction of the one-electron, low-redox- potential dye BV (11, 34, 51, 67, 101). The physiological electron acceptor of FDH-H has yet to be determined biochemically, although genetic evidence indicates that it is a protein encoded by the hyc operon (see below).

FDH-H is an 80-kDa selenopolypeptide and is encoded by the fdhF gene (11, 30, 34, 99, 159). Selenium, in the form of selenocysteine (SeCys), is located at amino acid position 140 in the FDH-H polypeptide chain (30, 134, 160). The FDH-Hpolypeptide was first purified in 1990 (11) and shown to contain 3.3 g atoms of iron and 1 g atom of molybdenum per mole of enzyme. These results suggested that the enzyme contains a single iron-sulfur cluster, and Heider and Böck (57) proposed that a conserved cysteine motif common to Mo-cofactor-dependent FDHs may be involved in forming a ligand to the cluster. Molybdenum was reported to be associated with the enzyme in the form of a molybdopterin guanine dinucleotide (MGD) cofactor (11).

Direct involvement of the selenolate of SeCys in formate oxidation was demonstrated by comparing the enzymic conversion of the selenocysteinyl enzyme with a cysteinyl-substituted derivative. The sulfur enzyme also proved to be 20 times less active than its selenium-containing counterpart at physiological pH, thus emphasizing the advantage of the reactivity of the selenol over the thiol group in redox processes (9, 10). A subsequent EPR spectroscopic analysis of ⁷⁷Se-enriched FDH-H revealed that the selenolate of the SeCys residue is directly coordinated to the molybdenum, which was suggested to be in the Mo(V) species (52). A more-detailed EPR analysis using near-homogeneous enzyme revealed that the molybdenum in formate-reduced, crystalline FDH-H was in the Mo(IV) oxidation state and that the single [4Fe-4S] cluster was reduced (51). Oxidation of the enzyme with BV generated the Mo(VI) species and an oxidized [4Fe-4S] cluster. This study gave the first insights into the reaction mechanism and the possible intramolecular electron transfer route, which were substantiated by the determination of the crystal structure of FDH-H (30).

 

Structure and Mechanism of FDH-H

The enzyme has a four-domain, αβ structure in which domain I coordinates the [4Fe-4S] cluster, domains II and III coordinate the 2 MGD in an αβα sandwich, and the C-terminal domain IV forms a cap over the two MGD cofactors (30). The active site is buried in a deep cleft and is formed from two MGDs, which coordinate the Mo atom through four cis-thiolates, and SeCys, which is directly coordinated to the Mo through the selenol. The sixth ligand to the Mo is a hydroxyl group of a water molecule and the ligands form a triangular prism pattern (Fig. 2A). The active site of the respiratory formate dehydrogenase, FDH-N (66), is almost identical with that of FDH-H and confirms that formate oxidation is carried out at the SeCys-coordinated Mo-bis-MGD cofactor. Indeed, despite the α-subunit of FDH-N being substantially larger than FDH-H, the core structure of both proteins is superimposable (66). The organization of the Mo-bis-MGD cofactor is a general framework conserved in this class of redox enzymes, facilitating intramolecular electron transfer (19, 65, 126).

A further feature of the FDH-H active site revealed by the structure is that the water molecule that is a ligand with the Mo also hydrogen bonds with the amide of His141 (30). Based on binding of the inhibitor nitrite, the substrate formate is suggested to be located between SeCys140 and His141 and this histidinyl residue is directly involved in proton abstraction. Moreover, unlike other Mo-bis-MGD-dependent molybdoenzymes, FDH-H releases CO₂ as product and, significantly, water is not the source of oxygen in formate oxidation. Upon oxidation the α-proton of formate is transferred to His141 via the SeCys residue, which undergoes transient protonation (Fig. 2B). This is also supported by EPR analysis (67).

Detailed kinetic studies using deuteroformate and proteoformate clearly demonstrated that the formate oxidation step is not rate limiting, but rather the subsequent one-electron transfer steps to BV in the in vitro analyses are rate determining (10). These findings have been substantiated by the structural data (30). The two electrons generated upon formate oxidation are transferred from the Mo(IV) to the [4Fe-4S] cluster, which is located just below the enzyme's surface and presumably transfers the electrons on to the next component in the FHL complex. The electrons are transferred one at a time, generating the Mo(V) species observed by EPR (51, 67). This enables SeCys to hydrogen bond to the amide of His141, thus stabilizing the protonated species. Further oxidation of Mo(V) to Mo(VI) results in breaking this SeCys-His141 hydrogen bond and releases the proton to the solvent.

The stoichiometry of FDH-H in the FHL complex is not known, mainly because the complex is extremely unstable and so has remained refractory to biochemical analysis. Future analyses must focus on elucidating the nature of the complete FHL complex.

 

Hydrogenase 3

Hyd-3 (EC 1.12.1.-) has proved even more recalcitrant to biochemical analysis than FDH-H. The existence of a third hydrogenase isoenzyme was first established by carrying out immunoprecipitation studies with antibodies raised against the Hyd-1 and Hyd-2 isoenzymes (123). The nonimmunoprecipitable hydrogenase enzyme activity could be correlated with FHL synthesis; however, activity was extremely labile and the enzyme could not be purified initially. The identification of the gene (hycE) encoding the large subunit of Hyd-3, which is located within the so-called hyc operon hycABCDEFGHI (see Fig. 1), facilitated subsequent characterization of the enzyme (29, 120). The HycE polypeptide has been purified and shown to contain up to 1 mol of nickel per mol of enzyme (113). Like FDH-H, the HycE polypeptide also appears to form a loose association with the other components of the FHL complex (120). It is likely that both of these polypeptides have an associated small subunit that functions in electron transfer within the complex (Table 2 and see below).

Five of the gene products of the hyc operon display significant sequence similarity with components of the NADH ubiquinone oxidoreductase (complex I) of the respiratory chain (29, 120). These are the proteins HycC, HycD, HycE, HycF, and HycG. They are related to the proteins NUO12, NUO8, NUO4, NUO9, and NUO2 of the bacterial complex I and with the proteins ND5, ND1, 49, TYKY, and PSST of the bovine complex I, respectively ( 29, 40, 152). Along with the fact that other subunits of the mammalian complex I share homology with components of the membrane-bound hydrogenases, this was taken as evidence that complex I and hydrogenases have diverged from a common ancestor (4, 47).

 

The Fourth Hydrogenase, Hyf

DNA sequence analysis identified a 12-cistron operon, termed hyf (hydrogenase four), on the E. coli chromosome that encodes a putative FHL-2 system (6). The operon (hyfABCDEFGHIRfocB; see Fig. 3 and Table 3) potentially codes for a hydrogenase complex comprising 10 subunits, resembling the FHL system. Two further genes within the operon encode a σ⁵⁴-dependent transcriptional regulator HyfR, which is related to the formate-responsive transcriptional regulator FHLA (128), and a putative formate channel, FocB, which is related to FocA (142). Seven of the putative subunits are orthologs of the seven Hyc subunits, while three subunits (HyfD, HyfE, and HyfF) have no related subunits in the Hyc complex. Rather, two of these three predicted integral membrane proteins (in addition to the five that are orthologs of the Hyc proteins) are related to subunits of the proton-translocating NADH:quinone oxidoreductase (complex I). It has been proposed that this 10-subunit complex functions together with FDH-H to form a novel proton-translocating system, which has been termed FHL-2 (6). Unfortunately, expression of the operon is very weak and it has not yet been possible to identify the gene products in wild-type E. coli cells (132, 133). One recent report, however, has suggested that the Hyf complex is responsible for dihydrogen production at pH 7.5 and that activity of the complex requires F₀F₁-ATPase (14). A further report was not able to substantiate this finding (132). Clearly, these observations require clarification, in particular, because hyf operon expression is weak. Nevertheless, clear evidence based on mutant analysis indicates that F₀F₁-ATPase is required for fermentative gas production in Salmonella (119).

 

REGULATION OF FORMATE AND HYDROGEN METABOLISM

 

Genetics of Hyd-1 and Hyd-2

Mutants specifically defective in Hyd-2 biosynthesis have been isolated (71, 74, 141). The mutations are likely to be located within the structural genes of the hyb operon, which encodes Hyd-2 (89, 104). The operon comprises eight genes located between 3138 and 3144 kbp on the E. coli chromosome (Table 4). The first four genes, hybO,A,B,C, encode structural components of the enzyme (118). The hybA gene was originally designated as the small subunit based on sequence homologies with the third subunit of the hydrogenase of Wolinella succinogenes (38). Amino acid sequence analysis of the purified Hyd-2 small subunit revealed that it is encoded by a previously unidentified gene, termed hybO, and this gene is located immediately upstream of hybA (118). Transcriptional studies confirmed the reassigned operon structure (107). The HybO protein has a 37-amino-acid signal sequence that has the characteristic RRXFXK signature of the Sec-independent Tat (twin arginine transport) pathway (18, 118). The fact that HybC lacks a signal sequence indicates that the HybC and HybO proteins fold and form a complex with bound cofactors prior to export to the periplasmic face of the membrane. Notably, the iron-sulfur protein HybA also has a Tat-signal sequence and it is likely that it is also transported to the periplasmic side of the membrane.

The hybD-G genes are essential for synthesis of a fully active Hyd-2 isoenzyme. HybD appears to be a protease required for maturation of HybC after nickel insertion (48, 113). HybE and HybF are both essential for Hyd-2 activity. HybF is functionally related to HypA and is required for Ni²⁺ delivery to the Hyd-1 and Hyd-2 (26, 60). HybG exhibits amino acid similarity with HypC and is required for Fe insertion and guiding the maturation machinery to Hyd-1 and Hyd-2 (27; see also below).

The structural genes of Hyd-1 are encoded by the hya operon located between 1031 and 1036 kbp on the E. coli chromosome (90, 91, 104). The hya operon has six genes with hyaA-C encoding the subunits of the enzyme (Table 4). The hyaC gene product exhibits homology with HybB and probably has a similar function. Like the accessory polypeptides encoded by the hyb operon, the hyaD-F gene products are essential for synthesis of functional Hyd-1 (91, 104). HyaD is an ortholog of HybD and is likely to be the specific protease required for maturation of Hyd-1 (48).

The large and small subunits of both Hyd-1 and Hyd-2 share extensive similarities with the respective hydrogenase polypeptides from other organisms, and the various implications these homologies may have with regard to the structure and function of hydrogenases in general have been reviewed in detail (46, 104, 146, 147, 155).

 

Regulation of hya and hyb Gene Expression

Only a few studies have been performed in which the regulation of hya and hyb operon expression has been examined. The findings from these studies, however, indicate that expression of both operons is complex (8, 32, 68, 107). Studies of enzyme levels indicate that Hyd-1 and Hyd-2 are anaerobically inducible (15, 123), and this has been confirmed by both transcript analysis (107) and the use of lacZ fusions (8, 32, 68, 107). The hya operon is induced 50-fold by anaerobiosis, and hyb operon expression is induced 10-fold after anaerobic growth on glucose and 20-fold when E. coli is grown on glycerol and fumarate. Although an fnr mutation was shown to reduce the levels of active Hyd-1 and Hyd-2 in anaerobically grown cells considerably, this was subsequently shown to be indirect and due to FNR-dependent control of nickel operon expression (108, 157). Expression studies confirmed that FNR control of hya and hyb is indirect and that, in the case of hya, the ArcA two-component transcriptional regulator (122) and AppY control anaerobic induction (8, 32, 68, 107). AppY appears to be part of a regulatory cascade and expression of the appY gene is negatively regulated by the two-component DpiAB system in aerobically grown cells (61). The nature of the AppY-binding site has not been determined.

Expression of hya also depends on the stationary phase sigma factor RpoS (8, 68), with operon expression being maximal in early-stationary phase. King and Pryzbyla (68) have also shown that hya is expressed maximally when the external pH of the growth medium is acidic and expression is abrogated under alkaline conditions. ArcA is required for pH regulation. Expression of hyb has the opposite response to external pH.

Precisely how hyb expression is controlled in response to anaerobic induction is unclear. Although anaerobic induction is reduced in an arcAfnr double null mutant, approximately 5-fold anaerobic induction is still evident (107). AppY is not involved in controlling hyb expression; however, cAMP-CRP appears to have an indirect effect on controlling expression. It is conceivable that cAMP-CRP controls expression of a regulatory gene that controls hyb operon expression directly.

Finally, anaerobic expression of both hya and hyb is reduced when nitrate is provided in the growth medium (107). The dual nitrate-responsive two-component systems NarXL and NarQP (140) are clearly involved in mediating nitrate repression. Nitrate repression of hyb operon expression can be accounted for solely through the NarXL and NarQP systems (107), although, surprisingly, anaerobic induction of hyb expression is abolished in a narP knock-out mutant. This suggests that NarL in the absence of NarP represses operon expression. Nitrate regulation of hya is more complex and, in the absence of both NarX and NarQ, nitrate repression is partially relieved but not to the extent that expression levels attain those observed in anaerobic glucose-grown cultures. This suggests the involvement of an additional system in mediating nitrate repression of hya expression. Clearly, much work still needs to be done to resolve the complexities of hya and hyb regulation.

 

THE FORMATE REGULON: fdhF, hyc, AND hyp

 

Genetics of FHL Complex Formation

An early genetic study resulted in the isolation of two mutants that were defective in FHL activity (100). One proved to carry a lesion in the fdhF gene, encoding the selenopolypeptide of FDH-H, while the other had an insertion element located within the second gene of a multicistronic hyc operon, encoding, among other polypeptides, the other structural components of the FHL complex (29, 99, 100, 158, 159). Further DNA sequence analysis around the hyc operon identified a large locus (Fig. 3) required for synthesis of active hydrogenase enzymes (29, 77, 128). The hyp operon encodes proteins involved in maturation of functional Hyd isoenzymes (see above).

Detailed in-frame deletion analysis and database searches (29, 120) have provided considerable information regarding the putative functions of the hyc operon gene products in the FHL complex (Table 2). The HycA polypeptide is not a structural component of the FHL complex but rather is a transcriptional regulator.

Transcription of FHL complex genes occurs only during fermentative growth conditions and is absolutely dependent on an acidic pH in the medium, formate, and the alternative sigma factor σ⁵⁴ (22, 23, 75, 76, 77, 112). Hence, the expression of the fdhF gene and the hyc and hyp operons is precisely coordinated. The isolation of trans-acting regulatory mutants identified the fhlA gene (Fig. 3, Fig. 4), which encoded the transcriptional regulator that coordinates the expression of these genes in the presence of a critical threshold level of formate (127, 128). The FHLA protein has similarity to regulators of two-component sensor-regulator pairs (128). It has been shown to bind specifically to a cis-regulatory sequence located approximately 100 bp upstream of the fdhF gene (129), previously characterized by deletion analysis to be essential for the formate-dependent expression of a fdhF-lacZ fusion (21). FHLA binds to two further cis-regulatory sequences; one sequence, termed IR1, is located between the hycA and hypA genes (see Fig. 5) of the divergently oriented hyc and hyp operons, while the second binding site (IR2) is located between the hycA and hycB genes (129). A further transcriptional unit comprising hydN and hypF is also regulated by FHLA (75, 83).

Studies using an in vitro coupled transcription–translation system have demonstrated that IR1 is necessary for activation of hyc operon transcription and IR2 is required to activate transcription of the hyp operon (59). Formate probably interacts with the N-terminal domain of the FHLA protein to effect transcriptional activation (70, 131). IHF has also been shown to be required to optimize the expression from this complex regulatory region and it has been proposed that one function may be to organize a supramolecular transcription complex (59). IHF is not involved in the transcriptional regulation of the fdhF gene.

Several of the hyp operon gene products are also required during respiratory growth conditions (see below). A FNR-dependent promoter is located within the hypAgene to ensure that sufficient levels of these proteins are present for the maturation of catalytically active hydrogenase isoenzymes, even in the absence of formate (77, 92). Although the original analysis of the hypA promoter suggested FNR-dependent activation occurs aerobically, a more recent reassessment indicates that transcription of the hypA promoter is FNR dependent anaerobically (92). It is unclear why this discrepancy exists; however, the latter finding is in accord with the requirement for the hyp gene products under fermentative growth conditions.

The fhlA gene is transcribed at a low level from its own promoter and this level is enhanced anaerobically through the activity of the FNR-dependent promoter within the hypA gene (see Fig. 5) (112). Activation of the FHLA-dependent promoter in front of hypA further increases fhlA gene transcription. This scenario presents a novel positive-feedback mechanism for transcriptional control of a regulon (75). The HycA protein appears to antagonize the action of FHLA, thus preventing persistent activation of the formate regulon (S. Hopper and A. Böck, unpublished data). Exactly how HycA achieves this is still unclear; however, it could function by direct modulation of FHLA activity through protein–protein interaction, or it could control the intracellular formate concentration, for example, by influencing the activity of formate channels such as FocA (142) or FocB (6).

 

The Formate Regulon

In the absence of formate, no transcription of any FHLA-dependent promoter occurs (112). This indicates that there is an absolute requirement for formate to interact with FHLA to enable transcriptional control of the regulon. Molybdate also has an important subsidiary role in control of formate regulon expression (111, 127; see below). Control of intracellular formate levels, therefore, determines whether the formate regulon is activated or not and this provides a simple mechanism that the cell uses to ensure that the FHL complex is only synthesized when it is required. Expression of the genes of the regulon is not activated when an alternative electron acceptor, such as nitrate, TMAO, or oxygen, is present or when the pH of the medium is above 7.

A model for control of the formate regulon (reviewed in reference 75) is presented in Fig. 5. When pyruvate formate-lyase (PFL) is activated under anaerobic conditions, formate, the product of the PFL reaction is excreted at neutral pH, via transport proteins, such as FocA (142). If no exogenous electron acceptor is available and as the external pH decreases, formate is transported back into the cell and once the intracellular concentration increases above the threshold, the regulon becomes activated. In the presence of alternative electron acceptors such as TMAO or nitrate, formate is metabolized in the periplasm by FDH-O and FDH-N (1, 112) and so formate does not accumulate intracellularly. Compartmentalization of formate determines whether the regulon is activated or not. A recent study has implicated the nitrate-responsive two-component NarXL component in mediating the nitrate effect by direct interaction of the NarL transcriptional regulator with the upstream regulatory elements of the FHLA-controlled genes (150). It should be pointed out, however, that in this study an appropriate control analysis in a fdn deletion mutant was not performed. Results of earlier studies demonstrated that preventing synthesis of alternative respiratory routes of formate metabolism, or introducing high levels of exogenous formate, relieve nitrate- and TMAO-dependent "repression" of the formate regulon (1, 112). These observations suggest that metabolic drainage of formate is the major mechanism underlying control of regulon induction.

Additional support for such a model comes from the enzymic properties of the two formate dehydrogenases and of FHLA, which compete for their substrate, formate, under anaerobic conditions. FDH-H has an apparent K_m for formate of about 26 mM (11); those of FDH-N (39) and FHLA (58) are 0.12 and 5 mM, respectively. Induction of formate-dependent nitrate respiration by nitrate results in the drainage of formate into the nitrate respiratory chain because of the superior K_m of FDH-N for the substrate formate. Levels required for induction of the FHL system are not reached.

Apart from being a member of the formate regulon, FHLA synthesis is also subject to translational control by OxyS, which is a 109-nucleotide-long, untranslated RNA induced under oxidative stress (5). OxyS RNA inhibits translation by pairing with the fhlA mRNA covering the ribosome-binding site plus a small region of the coding segment and forming a stable mRNA-antisense complex (7). It can be speculated that oxidative stress, which is characteristic of the lifestyle of E. coli, in this way can rapidly shut down translation of the components of the FHL system and thereby prevent wasteful synthesis of the oxygen-sensitive system.

 

Regulation of hyf Expression

Weak expression of a hyfA-lacZ fusion has been observed and this was shown to be FHLA and σ⁵⁴ dependent (133). Fermentative growth at low pH was required for expression and formate was shown to induce expression in a FHLA-dependent manner. Expression was also maximal after cultures had exited the exponential phase of growth, which correlates with a pH reduction and intracellular formate accumulation.

Transcription of the hyfR regulatory gene could not be detected in wild-type E. coli cells (132, 133). However, placing hyfR expression under the control of an inducible promoter revealed that HyfR could activate the hyf operon to significant levels. Furthermore, expression only occurred anaerobically and was formate independent. HyfR was not able to activate the formate regulon and this indicates that it has different properties to FHLA (132, 133). Skibinski et al. (133) made the interesting observation that HyfR has a -C-X₆-H-C-X-C-P-X-C-X-P- motif, which suggests that it might coordinate a metal center such as an Fe-S cluster.

The cAMP-CRP protein has also been shown to influence hyf expression, but this might be an indirect effect (132). It is necessary to identify the physiological conditions under which hyf is expressed and these may well provide clues as to the function of the operon gene products in fermentative metabolism.

 

EFFECTS OF METALS ON REGULATION OF FHL GENES

Iron, nickel, molybdenum, and selenium are essential for the assembly and maturation of functional FHL complex. Selenium is an essential component of FDH-H and is incorporated as selenocysteine (Chapter Selenocysteine). There is no evidence that selenium affects transcription of the fdhF gene in E. coli.

A Ni²⁺-specific transport system is encoded by the nikABCDE operon (93), and its expression is regulated by FNR and the nickel-responsive regulator, NikR (35, 156). Nickel is not known to regulate expression of the hyc or hyp genes directly.

Mutants unable to transport molybdate are impaired in expression of the fdhF and hyc genes (111, 127). This defect can be complemented phenotypically by addition of high levels of molybdate to the medium. A further mutational study identified the Mo-responsive transcriptional regulator ModE and the MoeA protein to be required for the effect of molybdate on hyc expression (56, 130). The biochemical function of MoeA is not clear but it is thought to provide an activated form of molybdate, which then interacts with ModE to regulate hyc transcription. Mo-dependent binding of ModE to the hyc promoter-regulatory region has been demonstrated and the presumptive ModE-Mo complex binds upstream of FHLA (130). The biochemical basis underlying transcriptional activation by ModE-Mo at the hyc promoter is unclear.

 

ASSEMBLY AND MATURATION OF THE HYDROGENASES

 

Genetic Analysis

Early work in a number of groups had shown that lesions in certain genes different from the structural genes block the generation of active hydrogenase enzymes. In E. coli, most of these genes were located in the 58 to 59 min region of the chromosome (74, 115, 116, 117, 151). Determination of the nucleotide sequence of this region (29, 76) and systematic knock-out of each gene by introducing an in-frame deletion revealed that this chromosome segment harbors the genes for the hydrogenase 3 component of the FHL system (hyc operon) (120) plus a total of six additional genes whose products are required for the synthesis of the active enzymes (77). They were designated hyp genes, since inactivation of most of them (hypB, hypD, hypE, and hypF) affected hydrogenase formation pleiotropically (62, 83). Exceptions were hypA and hypC, since they were only involved in the path to active hydrogenase 3; however, it was shown later that there are homologs of these genes in the operon coding for hydrogenase 2 (62, 89) and they fulfill the function of HypA and HypC in the formation of hydrogenases 1 and 2 (25, 26, 60).

Phenotypic features of mutants blocked in one of the hyp genes are, apart from the block in the formation of active enzyme, that the large subunit of the hydrogenase is accumulated in a precursor form containing a C-terminal extension, which is not present in the mature end product (62, 77). This extension, whose size is different in the precursors of the four hydrogenase large subunits, is removed during the maturation process. A second trait is that the precursor of the large subunits accumulated in each one of the hyp mutants does not contain nickel (83, 84). It was also shown that the mutation in one of the genes could be phenotypically suppressed by supplementation of the medium with nickel concentrations close to 1 mM (151). This indicated that the product, later on shown to be identical with HypB (84), is involved in nickel insertion.

Finally, an additional gene involved in hydrogenase maturation is encoded within the operon containing the structural genes for hydrogenases 1, 2, and 3 and is, therefore, cotranscribed with them. It is the gene encoding the endopeptidase; its product removes the C-terminal extension from the precursor of the large subunit (89, 91, 114).

 

Structure of the Metal Center

The X-ray structures of NiFe-hydrogenases from various biological sources have been determined and have revealed several common features that are relevant to our understanding of the maturation process (for review, see reference 45). The two subunits of the heterodimeric enzyme contact each other over an extraordinarily large interface and the NiFe metal center is located in the interior of the large subunit, close to the interface. The small subunit contains a specific number of FeS centers, which transfer the electrons to or from the catalytic NiFe metal center (41, 148, 149). The bimetallic center is coordinated within the protein via four cysteine thiolates derived from the large subunit, two of which function as bridging ligands. One pair of the cysteine residues is present in a strongly conserved motif located in the N-terminal domain of the large subunit, the second pair is present close to the C terminus of the matured subunit. As disclosed by infrared spectroscopy (12, 13, 55, 143) and X-ray analysis (148, 149), the Fe of the center contains three diatomic ligands: two are cyano groups and the third one is a carbonyl moiety (55, 102). Since structural information is not available for the metal center of any of the E. coli hydrogenases, all the data discussed here are interpreted using the information gained from the analysis of the enzyme from other organisms.

To elucidate their function in the hydrogenase maturation process, the hyp gene products were purified and their structural and functional properties were determined. Table 5 gives the characteristics of the individual components and lists the partial reactions in the maturation process in which they are involved. The scheme in Fig. 6 proposes the events taking place during maturation of the large subunit of hydrogenase 3 from E. coli, as far as they are known.

 

Biosynthesis of the Cyanide Ligands

Aerobic synthesis of cyanide by microorganisms is a well characterized reaction (28) and involves the participation of molecular oxygen as a substrate (69). Until recently, its formation under anaerobic conditions had not been described. Stimulated by the observation that HypF shares a sequence motif with O-carbamoyl transferases ( 44, 86, 98, 136), it was discovered that carbamoyl phosphate is required for the formation of active hydrogenases (97). Mutants with a lesion in carAB were devoid of activity and the defect could be rescued partially by the inclusion of citrulline as a source of carbamoyl phosphate in the medium (24, 97). The response to citrulline was augmented when its conversion to arginine was blocked by a mutation in the argG gene and by overproduction of the ornithine transcarbamoylase protein to overcome the unfavorable equilibrium (24). In a study conducted two decades ago, a screen for mutants of serovar Typhimurium lacking hydrogenase led to the isolation of pyrA mutants. Because of the lack of information on the hydrogenase active site at that time, the phenotype of these strains could not be interpreted in biochemical terms (17).

Purified HypF protein accepts carbamoyl phosphate as a substrate. In accordance with primary sequence similarities (135, 154) and structural properties (110) characteristic of acyl phosphatases, the HypF protein hydrolyzes carbamoyl phosphate in the absence of other substrates. In the presence of ATP, HypF catalyzes the carbamoyl phosphate-dependent cleavage of ATP into AMP and pyrophosphate (98). Inclusion of purified HypE protein in the reaction mixture showed that HypF carbamoylates the C-terminal cysteinyl residue of this protein, resulting in the generation of a protein–S-carboxamide (equation 1) (103). HypE then activates the oxygen of the carboxamide by ATP-dependent phosphorylation followed by dephosphorylation converting the protein-S-carboxamide into the protein-thiocyanate (equation 2). In summary, HypF functions as a carbamoyl transferase, while HypE catalyzes an ATP-dependent dehydratase reaction (103).

HypF + carbamoylphosphate + ATP + HypE-SH → HypF + HypE-S-carboxamide + AMP + PP_i + P_i (1)

HypE-S-carboxamide + ATP → HypE-thiocyanate + ADP + P_i (2)

It was postulated that the carbamoyl transfer to HypE involves the formation of a carbamoyl adenylate as an intermediate; however, this still needs experimental proof. Moreover, it remains to be determined whether carbamoyl phosphate also serves as the educt for the synthesis of the carbonyl ligand of the metal center. Two possibilities have been suggested, namely that HypE delivers the carbamoyl moiety to the iron where it is deaminated or that three cyanides are first attached to the iron from which one, via ligand chemistry, is hydrated and subsequently deaminated. It also cannot be excluded that CN and CO are formed from different chemical sources, as suggested by labeling experiments with Chromatium vinosum (102). If this is the case, either further gene product(s) must be involved or additional functions must be attributed to those of the currently characterized enzymes.

 

Transfer of the CN Ligand to the Iron

It is still unresolved at which site ligand formation of CN and CO to the iron takes place (Fig. 6). Does it take place on a "scaffolding" protein, as is the situation in the synthesis of certain Fe/S clusters (for review, see reference 43), or directly at the large hydrogenase subunit? Evidence for the former scenario was gained through the study of mutants devoid of carbamoyl phosphate synthetase activity in which the genes for Hyp proteins were overexpressed. A HypC-HypD complex accumulated and could be isolated by affinity chromatography. When cells harboring this complex were provided with citrulline as a source of carbamoyl phosphate, the complex was converted to a more slowly migrating species, but only when cells were devoid of the large hydrogenase subunit (24). Cells in which hydrogenase maturation was blocked at a later step, for example, nickel insertion or endoproteolytic processing, lacked the HypC-HypD complex but contained HypC in a complex with the precursor of the large subunit (37, 78). These results are interpreted in terms of a model in which HypC-HypD is the site of Fe-CN ligation and where the Fe carrying the ligands is subsequently transferred to the large subunit, possibly with HypC as a carrier (24). This assumption is supported by recent results, which show that the radioactivity of ¹⁴C-carbamoyl phosphate introduced onto HypE by HypF can be further transferred to the HypC-HypD complex isolated from anaerobically grown cells (M. Blokesch and A. Böck, unpublished results). It is intriguing that HypD carries a 4Fe-4S cluster with an apparently unusual coordination as judged by the absence of the classical sequence motifs involved in binding such clusters. Further work must address the question of whether one of the Fe atoms of this cluster is the target of ligand addition or whether there is an additional iron in the HypC-HypD complex, which serves as acceptor.

 

Nickel Insertion

There is convincing evidence that nickel insertion takes place at the precursor of the large hydrogenase subunit after iron incorporation. Arguments in favor of this are that the precursor present in cells grown under nickel limitation can be matured into active enzyme by the provision of nickel (81). Moreover, mutants with a deletion in any of the hyp genes contain a large subunit precursor devoid of nickel, whereas in a strain lacking the endopeptidase the large subunit contains the metal (84, 144).

Nickel insertion under physiological nickel concentrations in the medium requires the activity of two proteins, namely HypA and HypB (26, 62). HypB was first purified from E. coli (84) and later from other organisms (49, 88, 106) and shown to be a GTP-binding and -hydrolyzing protein. Indeed, mutant HypB proteins with amino acid replacements that interfere with hydrolysis of GTP are unable to incorporate nickel and mature the large subunit (85, 87, 88, 94). HypB from E. coli, therefore, is the first GTPase for which a role in metal insertion into a protein was discovered. HypA, on the other hand, is a nickel-binding protein, as was initially demonstrated for the protein from Helicobacter pylori (88, 95) and subsequently also for HybF from E. coli. HybF is a homolog of HypA and functions in the maturation of Hyd-1 and Hyd-2 (26, 60). Apart from binding nickel, HybF contains stably bound zinc, putatively coordinated in a classical zinc finger domain (26). A model proposing a mechanism for HypA and HypB function was recently presented. It suggests that HypA, or its homolog HybF, binds nickel and guides it to the target protein, while HypB via its GTPase activity acts as a switch to regulate the reversible association with and/or dissociation from the large subunit. Attempts to prove the model in an in vitro maturation system have so far failed, which suggests that some crucial component is lacking, or a particular condition of the reaction is not met (26).

As already mentioned, one class of mutants blocked in hydrogenase maturation can be rescued by high nickel in the medium (151). This also holds for strains with defective hypA or hybF genes, as well as for a mutant lacking active hypA, hypB, and hybF genes (26, 60, 95). This finding is substantiated by the demonstration that the precursor of the large subunit present in nickel-starved cells can be matured in vitro in an extract lacking either HypB or HypA protein by adding high concentrations of nickel. The conclusion is that nickel can be forced into the apoprotein in the absence of HypA or HypB; their requirement, therefore, is not absolute and they may increase the fidelity or the kinetics of metal incorporation.

 

Endoproteolytic Maturation of the Precursor

The last step in the maturation process involves the endoproteolytic removal of a short peptide from the C terminus of the hydrogenase large subunit precursor (53, 113; see also ref. 82). This only occurs after nickel has been inserted (79). The cleavage site is two amino acid residues C-terminal to cysteine-4, which acts as a bridging ligand in the coordination of the metal center. The endopeptidases responsible are substrate specific; E. coli possesses at least three of them, HyaD, HybD, and HycI with specificity for Hyd-1, Hyd-2, and Hyd-3, respectively (89, 91, 114). Investigation of Hyd-4 maturation has not been reported.

HybD and HycI have been purified and the proteolytic activity of HycI has been demonstrated in vitro (48, 81, 114). HybD could be crystallized with cadmium in the crystallization buffer. In the crystals of the protein, which has an α/β structure, one cadmium ion is bound by HybD within a cleft, possibly representing the active site. The cadmium is penta-coordinated by the carboxylate oxygens of a glutamic acid and an aspartic acid, the imidazole nitrogen of a histidine side chain, and a water molecule (48). Replacement of these residues by nonsimilar amino acids completely abolishes enzymic activity, whereas mutant proteins with chemically similar amino acid substitutions retain residual activity. This indicates that the role of these amino acids in metal coordination is essential and that the cadmium-binding site in the crystal is the proposed nickel-binding site in the native protein (144). Since purified HybD and HycI proteins are devoid of metal and also do not bind nickel tightly in vitro, it was concluded that the nickel present in the precursor of the large subunit is used as a recognition motif for the endopeptidase (80, 144). Indeed, whereas proteolytic processing of a nickel-free precursor of the large subunit is inhibited by addition of a nickel-complexing agent, this was not observed when nickel had already been incorporated.

What is the role of the proteolytic processing within the maturation cascade? An indication of the function may be taken from the fact that the precursor of the large subunit undergoes a dramatic change in its electrophoretic mobility in nondenaturing polyacrylamide gels after cleavage (144). The interpretation is that the extension holds the protein in a more open form, which can accept the metals, and that the removal of the C-terminal peptide induces a conformational change into a compact conformation with the metal center in the interior of the subunit (20). Evidence for such a role also comes from results of experiments in which the extension was truncated from the C-terminal end. Although processing was not grossly affected down to a critical lower limit in size, the overall stability of the total precursor was dramatically reduced. This indicates that the C-terminal extension interacts with the main body of the molecule, stabilizing it in a particular conformation (20, 145).

A comparison of cleavage sites from various precursors reveals a striking conservation of amino acids. The amino acid at position –1 is usually a histidine or an arginine, and at position +1 a nonpolar residue is found. Surprisingly, however, certain amino acid substitutions introduced into the precursor of Hyd-3 reveal a remarkable tolerance. Thus, exchange of the arginine by other basic or nonpolar residues did not influence cleavage; acidic and large nonpolar residues, however, yielded enzymically inactive matured subunit. Replacement of the methionine following the cleavage position by acidic residues abolished processing, whereas substitution by other nonpolar amino acids was tolerated. Again, the replacement by polar residues greatly destabilized the whole protein (145; Theodoratou and Böck, unpublished results). Altogether, one can conclude that the C-terminal extension may function as an intramolecular chaperone stabilizing the precursor in a conformation required for metal incorporation.

Figure 6gives a schematic overview of the path of maturation as predominantly worked out for hydrogenase 3 from E. coli. It is thought that, rather than functioning in a sequential manner, the reactions take place in multiprotein complexes. For example, a complex between proteins HypC-HypD-HypE could be isolated by affinity chromatography in which HypE accepts the carbamoyl residue from HypF and then transfers it to HypC-HypD (M. Blokesch and A. Böck, unpublished results). Moreover, an interaction between proteins HypA and HypB has been observed in the H. pylori maturation system, the two proteins possibly working as a "nickel insertase" (88).

Differences might also exist between the overall maturation process of the large subunit of Hyd-3 and that of Hyd-1 and Hyd-2. Hyd-3 is attached to the inner side of the cytoplasmic membrane (120), consequently the maturation takes place also in the absence of the small subunit (74). In contrast, Hyd-1 and Hyd-2 are transported to the periplasmic side of the membrane as the fully matured and assembled heterodimers (109). Therefore, the maturation of the large and small subunits may be interdependent (Chapter Protein Secretion and Targeting).

 

MATURATION NETWORK

The genetic control of synthesis of the FHL system is tightly linked to the process of fermentation, since formate is the master regulator. The regulation of formation of Hyd-1 and Hyd-2, on the other hand, although being more complex appears to be mainly connected with anaerobic respiration (see above). Epistatic to these genetic control mechanisms, however, there is interdependence between synthesis at the level of enzyme maturation and the activity of the orthologous pairs of maturation proteins HypA/HybF and HypC/HybG. HypA and HybF are nickel-binding proteins and they both require the activity of protein HypB for nickel insertion (26, 60, 62, 88, 95). HypC and HybG, on the other hand, both form a complex with protein HypD; this complex is an intermediate in the process of the attachment of the ligands to the iron of the NiFe center (25, 26). Thus, HypA and HypC guide the assembly process in the direction of Hyd-3, whereas HybG and HybF shift maturation in the direction of synthesis of Hyd-1 and Hyd-2 (Fig. 7). Under certain physiological conditions, therefore, the protein pairs must compete with each other. This can clearly be seen when one of the two partners is overproduced, resulting in a reduction in the maturation of the competing enzyme. Overproduction of HybG, for example, impairs the generation of mature Hyd-3 as it outcompetes HypC for the HypC-HypD complex and itself undergoes complex formation with HypD (24, 25). Under conditions where the FHL system and the hyp operon are fully induced by the formate generated during fermentation, the massive amount of HypC formed recruits HypD to Hyd-3 maturation, thus reducing the availability of HypD to interact with the HybG protein. .

 

HYDROGENASES IN OTHER ENTEROBACTERIA

Serovar Typhimurium has the FHL pathway, as well as Hyd-1 and Hyd-2 (63, 125). As with the E. coli enzyme, synthesis of Hyd-1 is induced at acid pH in serovar Typhimurium (96). However, in contrast to hya in E. coli, expression of the hya operon in serovar Typhimurium is absolutely dependent on the cAMP-CRP complex. Surprisingly, expression of hya in serovar Typhimurium has an absolute requirement for the tyrosine-dependent regulator TyrR. No dependence of E. coli hya on tyrosine has been reported.

As well as having a Ni²⁺-dependent FHL system, Klebsiella pneumoniae has a membrane-associated NAD(P)⁺-reducing hydrogenase activity (139). This enzyme is induced when K. pneumoniae grows fermentatively with citrate as a carbon source. Dihydrogen is an end product of citrate fermentation (31) and it has been proposed that the physiological role of the hydrogenase is to provide reduced pyridine nucleotides for biosynthesis (139). This is analogous to the role of hydrogen-recycling proposed for Hyd-1 in E. coli (123); however, in K. pneumoniae the hydrogenase has been shown to contribute directly to biomass formation (139). This is the first report of a pyridine nucleotide-reducing hydrogenase in enterobacteria.

 

PHYSIOLOGY OF Hyd-1 AND Hyd-2

Although Hyd-1 and Hyd-2 are present at substantial levels when E. coli or serovar Typhimurium cells ferment hexoses (63, 123, 124), it is still questionable whether they can be classified as true enzymes of fermentation. It is clear that Hyd-2 is the principle H₂-oxidizing activity when E. coli cells grow on dihydrogen and fumarate (123, 124) and the enzyme is probably proton translocating (64). Hyd-1 spans the cytoplasmic membrane and is probably also an energy-conserving enzyme (54). The likely topologies of the enzymes in the cytoplasmic membrane are also in agreement with their H₂-oxidizing capacities (Fig. 1).

In a recent study the capacity of E. coli to catalyze dihydrogen uptake in mutants deficient in Hyd-1 or Hyd-2 was examined (72). Hyd-2 coupled dihydrogen oxidation to reduction of electron acceptors with low midpoint potentials, while Hyd-1 was unable to couple to these acceptors. In contrast, Hyd-1 could couple dihydrogen oxidation to dioxygen reduction, a reaction that could not be catalyzed by Hyd-2 efficiently. These findings suggest that Hyd-1 allows E. coli to shuttle between aerobiosis and anaerobiosis by coupling dihydrogen oxidation to the reduction of high-potential electron acceptors, for example, nitrate and DMSO, while Hyd-2 is functional in the low redox potential range. This hypothesis was supported by more recent work in which dihydrogen oxidation was coupled to mediators of different redox potential (73). Taken together, these findings suggest that Hyd-2 functions optimally in the redox range below –80 mV, while Hyd-1 is optimal in the range +30 mV to +110 mV. If substantiated, these results would suggest that Hyd-1 and Hyd-2 provide complimentary dihydrogen oxidation activities, covering a range of redox potentials to which facultative anaerobes such as E. coli might be exposed.

The finding that Hyd-1 and Hyd-2 respond differentially to external pH also indicates a complimentary role of the two enzymes in anaerobic hydrogen metabolism (68) and is entirely consistent with the respective pH optima of the enzymes (16, 124). Furthermore, the strong response of hya expression to acidic pH and stationary phase point to a role of Hyd-1 in stress survival, perhaps through maintenance of the proton-motive force via energy-conserving dihydrogen oxidation (68).

It has been proposed that either Hyd-1 or Hyd-2, or both, could serve the function of recycling the dihydrogen evolved by the FHL complex during fermentation (123, 124). Such a H₂-recycling mechanism could be useful in facilitating redox balance, for example, when particularly reducing substrates such as sugar alcohols are oxidized in the absence of exogenous electron acceptors (33). Evidence has been presented which indicates that, when growing on sorbitol, fermenting E. coli cells produce excess ethanol and increased amounts of succinate relative to acetate and formate, which cannot be accounted for by calculating redox balance using standard fermentation pathways (3). One means of accounting for the excess ethanol production would be if the reducing equivalents from formate were recycled and channeled to fumarate via the quinone pool. This could be achieved by Hyd-1- or Hyd-2-dependent reoxidation of some of the dihydrogen produced by the FHL complex. Indeed, Alam and Clark (3) could show that in a hypB mutant, which is incapable of synthesizing any of the Hyd isoenzymes, the amount of ethanol and succinate produced was significantly decreased. Substantiation of these results using specific deletion mutants may strengthen the proposal that Hyd-1 and Hyd-2 can function in fermentation.

 

ACKNOWLEDGMENTS

A.B. received support from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie. Work in the laboratory of R.G.S. is supported by the BBSRC.

R. Harris is thanked for help in preparing the figures.