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.
Gross
Roland
R
Institut für Mikrobiologie, Johann Wolfgang Goethe-Universität, Marie-Curie-Str 9, Frankfurt am Main, Germany.
Pisa
René
R
Sänger
Monica
M
Lancaster
C Roy D
CR
Simon
Jörg
J
D019943:Amino Acid Substitution; D001483:Base Sequence; D001665:Binding Sites; D045303:Cytochromes b / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D006864:Hydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D024482:Vitamin K 2 / Q000378:metabolism*; D016968:Wolinella / Q000737:chemistry*
D017931:DNA Primers; D011994:Recombinant Proteins; D024482:Vitamin K 2; D045303:Cytochromes b; C043296:nickel-iron hydrogenase; D006864:Hydrogenase