The heme-copper oxidase superfamily: Heme-copper oxidase (HCO) and nitric oxide reductase (NOR) Heme-copper oxidases (HCOs) are a superfamily of terminal oxidases present in the respiratory chains of both bacteria and eukaryotic mitochondria.5 HCOs utilize O2 as a terminal electron acceptor, catalyzing the four electron reduction of O2 to H2O (O2 + 4e− + 8H+in → 2H2O + 4H+out), while generating a proton gradient that is used to drive the production of ATP. Nitric oxide reductase (NOR) is a metalloenzyme in the denitrification pathway responsible for the two electron reduction of NO to N2O (2NO + 2H+ + 2e− → N2O + H2O), a reaction that is nonelectrogenic (i.e., no charge translocation across the membrane).6,7 Denitrifying NO reductases are also members of the heme-copper oxidase superfamily. However, despite the diversity of their native functions, NOR and HCO have cross-reactivity; NOR is capable of reducing O2 and HCO is capable of reducing NO, although less efficiently.8–10 Not all members of the HCO superfamily are capable of NO reduction. The oxidase from bovine heart has no NO activity.11 In contrast, the ba3 and caa3 type oxidases from Thermus thermophilus9 and the cbb3 type oxidase from Pseudomonas stutzeri10 are capable of catalytically reducing NO to N2O, with the cbb3 oxidase being ~2 orders of magnitude faster than their oxidase counterparts. Although X-ray crystal structures are available5 for the oxidase from bovine heart,12,13 the aa3 type from Paracoccus denitrificans14,15 and Rhodobacter sphaeroides,16 the ba3 type from Thermus thermophilus,17 and the bo3 type ubiquinol oxidase from Escherichia coli,18 there is still no crystal structure for the cbb3 type oxidase, which from sequence alignments are predicted to be the most structurally similar oxidase to NOR.19 These crystal structures show the catalytic core of HCOs to contain a low-spin heme center and a high-spin heme/CuB binuclear center, (cytochrome c oxidase, CcO, also a member of the HCO superfamily, contains an additional binuclear CuA center). The CuB center consists of three strictly conserved His which bind a central Cu ion. One of these His is covalently attached to a strictly conserved Tyr to form a His-Tyr crosslink that is thought to play both a structural and functional role in O2 reduction.20 Another important characteristic of the heme-copper active site is the spin-coupled nature of the center in the oxidized or met form (i.e., CuII-FeIII) of the protein. Spin-coupling is thought to be provided by a bridging ligand between the two metal centers.21–25 Currently, no crystal or solution structure exists for bacterial NOR but sequence alignments and structural modeling have shown NORs to be structurally homologous to subunit I of HCOs.6,7 All six His metal ligands to the active site of HCO (two to the low-spin heme, one to the high-spin heme and three to CuB) are conserved in NOR at the same predicted positions in the 12 trans-membrane helices of HCO. The major structural difference between HCO and NOR is the replacement of a copper binding site (CuB) with a non-heme iron binding site (FeB) in NOR. Additionally, several conserved glutamates have been identified in the vicinity of the catalytic heme/non-heme FeB site of NOR which are predicted to play a role in iron binding and/or catalysis.26 Because of the importance of HCO to respiration, the structural similarities between NOR and HCO, and the importance of NOR to the denitrification cycle, it is important to reach a clear understanding of the structure and function of these vital enzymes. Biochemical, spectroscopic, and X-ray crystallographic studies of both HCOs12–15,27–37 and NORs38–49 are sometimes hampered by their size and complexity. Both are membrane-bound proteins containing several different metal cofactors, making their isolation difficult and spectroscopic characterization complicated (e.g., UV-vis and MCD spectra of the heme-copper center in HCO are masked by the low-spin heme center). These difficulties call upon the need for innovative ways to gain insight into the structure-function relationship of these heme proteins.