Binding of NO and CO to the d₁ heme of cd₁ nitrite reductase from Pseudomonas aeruginosa

The cd₁ nitrite reductase, a key enzyme in bacterial denitrification, catalyzes the one-electron reduction of nitrite to nitric oxide. The enzyme contains two redox centers, a c-type heine and a unique d₁ heme, which is a dioxoisobacteriochlorin. Nitric oxide, generated by this enzymatic pathway, if not removed from the medium, can bind to the ferrous d₁ cofactor with extremely high affinity and inhibit enzyme activity. In this paper, we report the resonance Raman investigation of the properties of nitric oxide and carbon monoxide binding to the d₁ site of the reduced enzyme. The Fe-ligand (Fe-NO and Fe-CO) stretching vibrational frequencies are unusually high in comparison to those of other ferrous heine complexes. The frequencies of the Fe-NO and N-O stretching modes appear at 585 and 1626 cm^-1, respectively, in the NO complex, while the frequencies of the Fe-CO and C-O stretching modes are at 563 and 1972 cm^-1, respectively, for the CO complex. Also, the widths (fwhm) of the Fe-CO and C-O stretching modes are smaller than those observed in the corresponding complexes of other heine proteins. The unusual spectroscopic characteristics of the d₁ cofactor are discussed in terms of both its unique electronic properties and the strongly polar distal environment around the iron-bound ligand. It is likely that the influence of a highly ruffled structure of heme d₁ on its electronic properties is the major factor causing anomalous Fe-ligand vibrational frequencies.