Epsilonproteobacterial hydroxylamine oxidoreductase (εHao): characterization of a ‘missing link’ in the multihaem cytochrome c family

Summary Members of the multihaem cytochrome c family such as pentahaem cytochrome c nitrite reductase (NrfA) or octahaem hydroxylamine oxidoreductase (Hao) are involved in various microbial respiratory electron transport chains. Some members of the Hao subfamily, here called εHao proteins, have been predicted from the genomes of nitrate/nitrite‐ammonifying bacteria that usually lack NrfA. Here, εHao proteins from the host‐associated Epsilonproteobacteria Campylobacter fetus and Campylobacter curvus and the deep‐sea hydrothermal vent bacteria Caminibacter mediatlanticus and Nautilia profundicola were purified as εHao‐maltose binding protein fusions produced in Wolinella succinogenes. All four proteins were able to catalyze reduction of nitrite (yielding ammonium) and hydroxylamine whereas hydroxylamine oxidation was negligible. The introduction of a tyrosine residue at a position known to cause covalent trimerization of Hao proteins did neither stimulate hydroxylamine oxidation nor generate the Hao‐typical absorbance maximum at 460 nm. In most cases, the εHao‐encoding gene haoA was situated downstream of haoC, which predicts a tetrahaem cytochrome c of the NapC/NrfH family. This suggested the formation of a membrane‐bound HaoCA assembly reminiscent of the menaquinol‐oxidizing NrfHA complex. The results indicate that εHao proteins form a subfamily of ammonifying cytochrome c nitrite reductases that represents a ‘missing link’ in the evolution of NrfA and Hao enzymes. This work reports the characterization of an octahaem multihaem cytochrome c (here named εHao since it is structurally related to hydroxylamine oxidoreductases) that occurs in various environmentally important organisms, most of which are Epsilonproteobacteria. Four prototypic εHao enzymes were purified and shown to be competent ammonium‐forming nitrite and hydroxylamine reductases, thus functionally resembling pentahaem cytochrome c nitrite reductase (NrfA). Consequently, εHao proteins represent a missing link within the ever‐growing multihaem cytochrome c family.