Evolution of an octahaem cytochrome c protein family that is key to aerobic and anaerobic ammonia oxidation by bacteria

Abstract

The biogeochemical nitrogen cycle is mediated by many groups of microorganisms that harbour octahaem cytochromes c (OCC). In this study molecular evolutionary analyses and the conservation of predicted functional residues and secondary structure were employed to investigate the descent of OCC proteins related to hydroxylamine oxidoreductase (HAO) and hydrazine oxidoreductase (HZO) from pentahaem cytochrome c nitrite reductase (NrfA). An octahaem cytochrome cnitrite reductase (ONR) was shown to be a possible intermediate in the process. Analysis of genomic neighbourhoods of OCC protein-encoding genes revealed adjacent conserved genes whose products, together with HAO, provide a path of electron transfer to quinone and constitute a functional catabolic module. The latter has evolved more than once under a variety of functional pressures on the catabolic lifestyles of their bacterial hosts. Structurally, the archetypical long helices in the large C-terminal domain of the proteins as well as the distal axial ligands to most haems were highly conserved in NrfA and all descendents. Residues known to be involved in the nitrite reductase activity of NrfA including the ‘CxxCK’ motif at the catalytic haem, the substrate and Ca binding sites, and the nitrite and ammonium channels were conserved in the eight representatives of ONR. In the latter, a unique cysteine has been inserted above the active site. The 64 other OCC proteins differed from ONR by the absence of the ‘CxxCK’ motif, the channel residues and most of the Ca-binding residues and the conserved presence of an ‘Asp–His’ pair inserted above the active site as well as the tyrosine that forms an intersubunit cross-link to the catalytic haem of HAO. Our proposed scenario of evolution of OCC proteins in the HAO family from NrfA is supported by (i) homology based on sequence and structure, (ii) its wide distribution among bacterial taxa, (iii) the dedicated interaction with specific proteins, and it is (iv) congruent with geological history.