ON THE MECHANISM OF INHIBITION OF CYTOCHROME-C-OXIDASE BY NITRIC-OXIDE

The mechanism of inhibition of cytochrome (cyt) c oxidase by nitric ox ide (NO) has been investigated by stopped flow transient spectroscopy and singular value decomposition analysis, Following the time course o f cyt c oxidation at different O-2/NO ratios, we observed that the ons et of inhibition: (i) is fast and at a high NO concentration is comple te during the first turnover; (ii) is sensitive to the O-2/NO ratio; a nd (iii) is independent of incubation time of the oxidized enzyme with NO, Analysis of the reaction kinetics and computer simulations suppor t the conclusion that inhibition occurs via binding of NO to a turnove r 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 binucl ear center, since recovery of activity occurs at the rate of NO dissoc iation (k = 0.13 s(-1)), as determined in the absence of O-2 using hem oglobin as a NO scavenger, We propose that removal of NO from the medi um is associated with reactivation of the enzyme via a relatively fast thermal dissociation of NO from the reduced cyt a(3)-Cu-B center.