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The products of a minimum of 15 genes are required for the synthesis of an active formate-hydrogenlyase (FHL) system in Escherichia coli. All are co-ordinately regulated in response to variations in the oxygen and nitrate concentration and the pH of the culture medium. Formate is obligately required for transcriptional activation of these genes. Analysis of the transcription of one of these genes, hycB linked to the lacZ reporter gene, revealed that oxygen and nitrate repression of transcription could be relieved completely, or partially in the case of nitrate, either by the addition of formate to the medium or by increasing the copy number of the gene encoding the transcriptional activator (fhlA) of this regulon. These studies uncovered a further level of regulation in which the transcription of hycB was reduced in cells grown on glucose. This effect was most clearly seen in aerobically grown cells when formate was added externally. Addition of cAMP overcame this glucose repression, which could be shown to be mediated by the cAMP receptor protein. These results would be consistent with the transport of formate being regulated by catabolite repression. Moreover, the repression of transcription through high pH also could be partially overcome by addition of increasing concentrations of formate to the medium, again being consistent with regulation at the level of formate import and export. Taken together, all these observations indicate that it is the intracellular level of formate that determines the transcription of the genes of the formate regulon by FhlA. This represents a novel positive feedback mechanism in which the activator of a regulon induces its own synthesis in response to increases in the concentration of the catabolic substrate, and this in turn is governed by the relative affinities of FhlA and the three formate dehydrogenase isoenzymes for formate.

The large subunits of nickel-containing hydrogenases are synthesised in a precursor form which, after nickel incorporation, is processed by proteolytic cleavage at the C-terminal end. The protease involved in processing of HycE, the large subunit of hydrogenase 3 from Escherichia coli, was purified by three chromatographic steps to apparent homogeneity. Its gene was identified by using a hybridisation probe generated by PCR with oligonucleotide primers the sequence of which was derived from the N-terminal and internal amino acid sequences. Determination of the nucleotide sequence showed that the gene is located distally and as a hitherto uncharacterised gene within the hyc operon, coding for hydrogenase 3 components. It was designated hycl. The HycI protease has a molecular mass of 17 k Da and is a monomer. Its cleavage reaction is not inhibited by conventional inhibitors of serine and metalloproteases, which correlates with the fact that the sequence does not contain signature motifs characteristic of serine-, metallo-, cysteine-or acid proteases. Homologous genes are present in other transcriptional units coding for hydrogenases.Keywords. Protease HycI; carboxy-terminal processing ; [NiFeIhydrogenase; Escherichia coli.Escherichia coli possesses three nickel-dependent hydrogenases [l]. Hydrogenase 1 and 2 are uptake hydrogenases involved in anaerobic H, oxidation with the concomitant reduction of an electron acceptor like fumarate; hydrogenase 3 is a part of the formate hydrogenlyase complex which functions in hydrogen evolution during fermentation (for review see [2]). Genes coding for the constituent subunits of these three enzymes are located at three different sites on the E. coli chromosome and are clustered with four (hydrogenase 1 ; hya operon), five (hydrogenase 2; hyb operon) or six (hydrogenase 3; hyc operon) additional genes which code for redox carriers or for proteins of unknown function [3-51. Mutations in most of the hya, hyb and hyc genes from E. coli specifically affect the formation of active hydrogenases 1, 2 and 3, respectively [4, 6, 71. Mutations in other genes (hyp), however, coding outside these three operons block the formation of all three hydrogenases pleiotropically [8, 91, i.e. they appear to have a general function in hydrogenase formation. Possibilities that were considered are folding, metal incorporation or membrane integration. Mutations in several genes of the hya, hyb and hyc operon and inactivation of the hyp genes lead to the accumulation of the large subunits of the hydrogenases in a Cterminally extended form [6,7, 91. This precursor of the large subunit was shown to be present also in several hydrogenase- deficient mutants of other organisms [lo-131 and, in addition, when the cells were starved for nickel [14]. It was amenable to in vitro processing provided that nickel had already been incorporated [15] or that nickel was present in the processing mixture [14]. Processing takes place at a histidine [16-181 or arginine [15] residue three amino acids C-terminal to a cy...

Purification of the large subunit, HYCE, of Escherichia coli hydrogenase 3 revealed that it is a nickel-containing polypeptide, which is subject to C-terminal proteolytic processing. This processing reaction could be performed in vitro with partially purified components, yielding a low-molecular mass C-terminal peptide which was resolved in a Tricine/SDS/polyacrylamide gel. N-terminal sequencing of this peptide revealed that proteolytic cleavage occurred at the C-terminal side of the arginine residue at position 537, which corresponds to the histidine residue in the highly conserved motif, DPCXXCXXH, of other (NiFe) hydrogenases thought to be involved in active site nickel coordination. Nickel-containing HYCE precursor for in vitro processing, was partially purified from strain HD708 (AhycH) in the presence of the reducing agent dithiothreitol. Using 2-mercaptoethanol instead of dithiothreitol provided pure precursor, which was, however, no longer susceptible to in vitro processing ; it proved to be devoid of nickel indicating that nickel incorporation into the HYCE precursor is a prerequisite for processing. This conclusion was supported by the finding that HYCE precursor from strain HD708 (AhycH) chromatographed with radioactivity from 63Ni incorporated in vivo and could be processed in vitro, whereas HYCE precursor from strain BEF314 (AhypB-E) lacking the nickel insertion system appeared to be devoid of nickel and was not sensitive to in vitro processing.According to their different physiological roles in bacterial metabolism, nickel-containing hydrogenases form a very heterogeneous group of enzymes. They differ with respect to their cellular function (H, oxidation or H, production), metal content, subcellular localization, subunit composition and the electron acceptors and donors used [l]. However, one subunit, called the large subunit, is common to all of these hydrogenases. In the amino acid sequence, it is the most conserved subunit and it is thought to ligand the active-site nickel. Two highly conserved cysteine-rich motifs are present in all known large subunits and probably provide the ligands required for the binding of nickel. One motif, RXCXXC, is located in the N-terminal region and the second, DPCXXCXXH, is located at the C-terminus of the polypeptide chain [l, 21. In addition to the genes encoding the hydrogenase subunits, a number of genes have been sequenced which are required for the formation of active hydrogenases [ 3 -101. Such activation of (NiFe) hydrogenases may involve insertion of nickel into the hydrogenase apoprotein [ l l -131, activation of nickel in the active site, membrane integration [3, 141, complex formation, processing of the small subunit carrying a signal peptide at its N-terminus [15] and processing of the large subunit [3,16, 171 which order these processes take place and whether further reactions are involved.The facultative anaerobe Escherichia coli possesses three hydrogenase isoenzymes [ 181. Two of these isoenzymes (hydrogenases 1 and 2) are membrane-bound uptake hy...

This work addresses the biogenesis of heme-copper terminal oxidases in Bradyrhizobium japonicum, the nitrogen-fixing root nodule symbiont of soybean. B. japonicum has four quinol oxidases and four cytochrome oxidases. The latter include the aa 3 -and cbb 3 -type oxidases. Although both have a Cu B center in subunit I, the subunit II proteins differ in having either a Cu A center (in aa 3 ) or a covalently bound heme c (in cbb 3 ). Two biogenesis factors were genetically studied here, the periplasmically exposed CoxG and ScoI proteins, which are the respective homologs of the mitochondrial copper-trafficking chaperones Cox11 and Sco1 for the formation of the Cu B center in subunit I and the Cu A center in subunit II of cytochrome aa 3 . We could demonstrate copper binding to ScoI in vitro, a process for which the thiols of cysteine residues 74 and 78 in the ScoI polypeptide were shown to be essential. Knock-out mutations in the B. japonicum coxG and scoI genes led to loss of cytochrome aa 3 assembly and activity in the cytoplasmic membrane, whereas the cbb 3 -type cytochrome oxidase apparently remained unaffected. This suggests that subunit I of the cbb 3 -type oxidase obtains its copper cofactor via a different pathway than cytochrome aa 3 . In contrast to the coxG mutation, the scoI mutation caused a decreased symbiotic nitrogen fixation activity. We hypothesize that a periplasmic B. japonicum protein other than any of the identified Cu A proteins depends on ScoI and is required for an effective symbiosis.