Modification of heme c binding motifs in the small subunit (NrfH) of the Wolinella succinogenes cytochrome c nitrite reductase complex.

The two multiheme c-type cytochromes NrfH and NrfA form a membrane-bound complex that catalyzes menaquinol oxidation by nitrite during respiratory nitrite ammonification of Wolinella succinogenes. Each cysteine residue of the four NrfH heme c binding motifs was individually replaced by serine. Of the resulting eight W. succinogenes mutants, only one is able to grow by nitrite respiration although its electron transport activity from formate to nitrite is decreased. NrfH from this mutant was shown by matrix-assisted laser desorption/ionization mass spectrometry to carry four covalently bound heme groups like wild-type NrfH indicating that the cytochrome c biogenesis system II organism W. succinogenes is able to attach heme to an SXXCH motif.

The nrfI gene is essential for the attachment of the active site haem group of Wolinella succinogenes cytochrome c nitrite reductase.

The cytochrome c nitrite reductase complex (NrfHA) is the terminal enzyme in the electron transport chain catalysing nitrite respiration of Wolinella succinogenes. The catalytic subunit NrfA is a pentahaem cytochrome c containing an active site haem group which is covalently bound via the cysteine residues of a unique CWTCK motif. The lysine residue serves as the axial ligand of the haem iron. The other four haem groups of NrfA are bound by conventional haem-binding motifs (CXXCH). The nrfHAIJ locus was restored on the genome of the W. succinogenes DeltanrfAIJ deletion mutant by integration of a plasmid, thus enabling the expression of modified alleles of nrfA and nrfI. A mutant (K134H) was constructed which contained a nrfA gene encoding a CWTCH motif instead of CWTCK. NrfA of strain K134H was found to be synthesized with five bound haem groups, as judged by matrix-assisted laser-desorption/ionization (MALDI) mass spectrometry. Its nitrite reduction activity with reduced benzyl viologen was 40% of the wild-type activity. Ammonia was formed as the only product of nitrite reduction. The mutant did not grow by nitrite respiration and its electron transport activity from formate to nitrite was 5% of that of the wild-type strain. The predicted nrfI gene product is similar to proteins involved in system II cytochrome c biogenesis. A mutant of W. succinogenes (stopI) was constructed that contained a nrfHAIJ gene cluster with the nrfI codons 47 and 48 altered to stop codons. The NrfA protein of this mutant did not catalyse nitrite reduction and lacked the active site haem group, whereas the other four haem groups were present. Mutant (K134H/stopI) which contained the K134H modification in NrfA in addition to the inactivated nrfI gene had essentially the same properties as strain K134H. NrfA from strain K134H/stopI contained five haem groups. It is concluded that NrfI is involved in haem attachment to the CWTCK motif in NrfA but not to any of the CXXCH motifs. The nrfI gene is obviously dispensable for maturation of a modified NrfA protein containing a CWTCH motif instead of CWTCK. Therefore, NrfI might function as a specific haem lyase that recognizes the active site lysine residue of NrfA. A similar role has been proposed for NrfE, F and G of Escherichia coli, although these proteins share no overall sequence similarity to NrfI and belong to system I cytochrome c biogenesis, which differs fundamentally from system II.

Characterization of the menaquinone reduction site in the diheme cytochrome b membrane anchor of Wolinella succinogenes NiFe-hydrogenase.

The majority of bacterial membrane-bound NiFe-hydrogenases and formate dehydrogenases have homologous membrane-integral cytochrome b subunits. The prototypic NiFe-hydrogenase of Wolinella succinogenes (HydABC complex) catalyzes H2 oxidation by menaquinone during anaerobic respiration and contains a membrane-integral cytochrome b subunit (HydC) that carries the menaquinone reduction site. Using the crystal structure of the homologous FdnI subunit of Escherichia coli formate dehydrogenase-N as a model, the HydC protein was modified to examine residues thought to be involved in menaquinone binding. Variant HydABC complexes were produced in W. succinogenes, and several conserved HydC residues were identified that are essential for growth with H2 as electron donor and for quinone reduction by H2. Modification of HydC with a C-terminal Strep-tag II enabled one-step purification of the HydABC complex by Strep-Tactin affinity chromatography. The tagged HydC, separated from HydAB by isoelectric focusing, was shown to contain 1.9 mol of heme b/mol of HydC demonstrating that HydC ligates both heme b groups. The four histidine residues predicted as axial heme b ligands were individually replaced by alanine in Strep-tagged HydC. Replacement of either histidine ligand of the heme b group proximal to HydAB led to HydABC preparations that contained only one heme b group. This remaining heme b could be completely reduced by quinone supporting the view that the menaquinone reduction site is located near the distal heme b group. The results indicate that both heme b groups are involved in electron transport and that the architecture of the menaquinone reduction site near the cytoplasmic side of the membrane is similar to that proposed for E. coli FdnI.