Nitric oxide reductase is a cytochrome bc complex that exhibits in the primary structure of the cytochrome b subunit (NorB) homology to the catalytic subunit of the heme-copper oxidase family. Based on this homology an atomic model for NorB from P. stutzeri was generated, using the crystal structure of the subunit I of cytochrome c oxidase from P. denitrificans. Most parts of the 12 transmembrane helices of NorB are superimposable on the COX I structure. Optical and magnetic circular dichroism spectra were analyzed to differentiate the heme-binding sites of the NO reductase complex. The room temperature MCD data clearly indicate three distinct heme species, present at comparable levels. Two of these are low-spin ferric hemes with histidine-methionine and bis-histidine coordination in NorC and NorB, respectively. The third is a high-spin ferric heme (NorB) with properties suggestive of histidine and an as yet unidentified anion as axial ligands. This high-spin heme is proposed to be part of the binuclear heme and nonheme Fe catalytic site of the enzyme, analogous to the heme a₃- CuB binuclear site. Our model is supported by phoA fusions and site-directed mutagenesis of His-258 which is proposed as a ligand to the nonheme Fe center.