Epsilonproteobacterial hydroxylamine oxidoreductase ( ε Hao): 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 ε Hao proteins, have been predicted from the genomes of nitrate/nitrite‐ammonifying bacteria that usually lack NrfA. Here, ε Hao 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 ε Hao‐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 ε Hao‐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 ε Hao proteins form a subfamily of ammonifying cytochrome c nitrite reductases that represents a ‘missing link’ in the evolution of NrfA and Hao enzymes.