Two CO molecules can bind concomitantly at the diiron site of NO reductase from Bacillus azotoformans.

CO complexes formed in reduced nitric oxide reductase from Bacillus azotoformans were investigated with resonance Raman and FTIR techniques. These experiments shows the presence of two nu(C-O) bands, one at approximately 1970 cm-1 assigned to the heme-CO complex, and one at approximately 2070 cm-1 from the non-heme iron, FeBCO. At cryogenic temperatures, the heme-CO complex adopts a semi-bridging configuration with FeB which decreases its stretching frequency to approximately 1910 cm-1 and decreases the nu(C-O) of FeBCO by approximately 20 cm-1. The concomitant binding of two CO molecules, one per iron(II) at the active site, is consistent with the formation of a [{FeNO}7]2 iron-nitrosyl dimer during substrate turnover. This study strongly supports the notion that this family of enzymes utilizes a reaction mechanism based on catalysis by proximity, where the formation of two iron-nitrosyl groups promotes N-N bond formation.