A common mechanism for the interaction of nitric oxide with the oxidized binuclear centre and oxygen intermediates of cytochrome c oxidase

The reactions of nitric oxide (NO) with fully oxidized cytochrome c oxidase (O) and the intermediates P and F have been investigated by optical spectroscopy, using both static and kinetic methods. The reaction of NO with O leads to a rapid (similar to 100 s(-1)) electron ejection from the binuclear center to cytochrome a and Cu-A. The reaction with the intermediates P and F leads to the depletion of these species in slower reactions, yielding the fully oxidized enzyme. The fastest optical change, however, takes place within the dead time of the stopped-flow apparatus (similar to 1 ms), and corresponds to the formation of the F intermediate (580 nm) upon reaction of NO with a species that we postulate is at the peroxide oxidation level. This species can be formulated as either Fe5+ = O Cu-B(2+) or Fe4+ = O Cu-B(3+), and it is spectrally distinct from the P intermediate (607 nm). All of these reactions have been rationalized through a mechanism in which NO reacts with Cu-B(2+), generating the nitrosonium species Cu-B(1+) NO+, which upon hydration yields nitrous acid and Cu-B(1+). This is followed by redox equilibration of Cu-B with Fe-a/Cu-A or Fe-a3 (in which Fe-a and Fe-a3 are the iron centers of cytochromes a and a(3), respectively). In agreement with this hypothesis, our results indicate that nitrite is rapidly formed within the binuclear center following the addition of NO to the three species tested (O, P, and F). This work suggests that nitrosylation at Cu-B(2+) instead of at Fe-a3(2+) is a key event in the fast inhibition of cytochrome c oxidase by NO.