Resonance Raman spectroscopy of nitric oxide reductase and cbb3 heme-copper oxidase

Elucidating the structure and properties of the active sites in cbb(3) heme-copper oxidase and in nitric oxide reductase (Nor) is crucial in understanding the reaction mechanisms of oxygen and nitric oxide reduction by both enzymes. In the work here, we have applied resonance Raman (RR) spectroscopy to investigate the structure and properties of the binuclear heme b(3)-Cu-B center of cbb(3) heme-copper oxidase from Pseudomonas stutzeri and the dinuclear heme b(3)-Fe-B center of Nor from Paracoccus denitrificans in the ligand-free and CO-bound forms and in the reactions with O-2 and NO. The RR data demonstrate that in the Nor/NO reaction, the formation of the N-N bond occurs with the His-Fe heme b(3) bond intact, and reformation of the heme b(3)-O-Fe-B dinuclear center causes the rupture of the proximal His-Fe heme b(3) bond. In the reactions of Nor and cbb(3) with O-2, distinct oxidized heme b(3) species, which differ from the as-isolated oxidized forms, have been characterized. The activation and reduction of O-2 and NO by cbb(3) oxidase and nitric oxide reductase are compared and discussed.