Conserved evolutionary units in the heme-copper oxidase superfamily revealed by novel homologous protein families

The heme-copper oxidase (HCO) superfamily includes HCOs in aerobic respiratory chains and nitric oxide reductases (NORs) in the denitrification pathway. The HCO/NOR catalytic subunit has a core structure consisting of 12 transmembrane helices (TMHs) arranged in three-fold rotational pseudosymmetry, with six conserved histidines for heme and metal binding. Using sensitive sequence similarity searches, we detected a number of novel HCO/NOR homologs and named them HCO Homology (HCOH) proteins. Several HCOH families possess only four TMHs that exhibit the most pronounced similarity to the last four TMHs (TMHs 9–12) of HCOs/NORs. Encoded by independent genes, four-TMH HCOH proteins represent a single evolutionary unit (EU) that relates to each of the three homologous EUs of HCOs/NORs comprising TMHs 1–4, TMHs 5–8, and TMHs 9–12. Single-EU HCOH proteins could form homotrimers or heterotrimers to maintain the general structure and ligand-binding sites defined by the HCO/NOR catalytic subunit fold. The remaining HCOH families, including NnrS, have 12-TMHs and three EUs. Most three-EU HCOH proteins possess two conserved histidines and could bind a single heme. Limited experimental studies and genomic context analysis suggest that many HCOH proteins could function in the denitrification pathway and in detoxification of reactive molecules such as nitric oxide. HCO/NOR catalytic subunits exhibit remarkable structural similarity to the homotrimers of MAPEG (membrane-associated proteins in eicosanoid and glutathione metabolism) proteins. Gene duplication, fusion, and fission likely play important roles in the evolution of HCOs/NORs and HCOH proteins.