On the Mechanism of Inhibition of Cytochrome c Oxidase by Nitric Oxide
Alessandro Giuffre, Paolo Sarti, Emilio D'Itri, Gerhard Buse, Tewfik Souliman
and Maurizio Brunori
J. Biol Chem. 271: 33404-33408, 1996
SUMMARY: The mechanism of inhibition of cytochrome c oxidase by NO has been
investigated by stopped-flow spectroscopy and singular value decomposition analysis.
Following the time course of cyt. c oxidation at different O2/NO ratios, we observed that
the onset of inhibition: (i) is fast and at at high [NO] is complete during the first
turnover; (ii) is sensitive to the O2/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 (preferential) binding of NO
to a turnover intermediate with a partially reduced cyt. a3-CuB binuclear center.
The inhibited enzyme has the optical spectrum typical of NO bound to reduced cyt. a3.
Reversal of inhibition in the presence of O2 does not involve a direct reaction of
O2 with NO while bound at the binuclear center, since recovery of activity occurs at
the rate of NO dissociation (0.13 sec-1), as determined in the absence of NO using hemoglobin
as a NO scavenger. We propose that removal of NO from the medium is associated with the
reactivity of the enzyme via a relatively fast thermal dissociation of NO from the
reduced a3-CuB center (followed b reaction with O2).