Reaction of nitric oxide with the oxidized di-heme and heme-copper oxygen-reducing centers of terminal oxidases: Different reaction pathways and end-products.

Inhibition of terminal oxidases by nitric oxide (NO) has been extensively investigated as it plays a role in regulation of cellular respiration and pathophysiology. Cytochrome bd is a tri-heme (b(558), b(595), d) bacterial oxidase containing no copper that couples electron transfer from quinol to O(2) (to produce H(2)O) with generation of a transmembrane protonmotive force. In this work, we investigated by stopped-flow absorption spectroscopy the reaction of NO with Escherichia coli cytochrome bd in the fully oxidized (O) state. We show that under anaerobic conditions, the O state of the enzyme binds NO at heme d with second-order rate constant k(on)=1.5+/-0.2x10(2) M(-1) s(-1), yielding a nitrosyl adduct (d(3+)-NO or d(2+)-NO(+)) with characteristic optical features (an absorption increase at 639nm and a red shift of the Soret band). The reaction mechanism is remarkably different from that of O cytochrome c oxidase in which the heme-copper binuclear center reacts with NO approximately three orders of magnitude faster, forming nitrite. The data allow us to conclude that in the reaction of NO with terminal oxidases in the O state, Cu(B) is indispensable for rapid oxidation of NO into nitrite.