On the mechanism of inhibition of cytochrome c oxidase by nitric oxide

The mechanism of inhibition of cytochrome (cyt) c oxidase by nitric oxide (NO) has been investigated by stopped flow transient spectroscopy and singular value decomposition analysis, Following the time course of cyt c oxidation at different O-2/NO ratios, we observed that the onset of inhibition: (i) is fast and at a high NO concentration is complete during the first turnover; (ii) is sensitive to the O-2/NO ratio; and (iii) is independent of incubation time of the oxidized enzyme with NO, Analysis of the reaction kinetics and computer simulations support the conclusion that inhibition occurs via binding of NO to a turnover intermediate with a partially reduced cyt a(3)-Cu-B binuclear center, The inhibited enzyme has the optical spectrum typical of NO bound to reduced cyt a(3). Reversal of inhibition in the presence of O-2 does not involve a direct reaction of O-2 with NO while bound at the binuclear center, since recovery of activity occurs at the rate of NO dissociation (k = 0.13 s(-1)), as determined in the absence of O-2 using hemoglobin as a NO scavenger, We propose that removal of NO from the medium is associated with reactivation of the enzyme via a relatively fast thermal dissociation of NO from the reduced cyt a(3)-Cu-B center.