The reaction of nitric oxide (NO) with oxidized fast cytochrome c oxidase w
as investigated by stopped-flow, amperometry, and EPR, using the enzyme as
prepared or after "pulsing," A rapid reduction of cytochrome a is observed
with the pulsed, but not with the enzyme as prepared. The reactive species
(lambda(max) = 424 nm) reacts with NO at k = 2.2 x 10(5) M-1 s(-1) at 20 de
grees C and is stable for hours unless Cl- is added, in which ease it decay
s slowly (t(1/2) similar to 70 min) to an unreactive state (lambda(max) = 4
23 nm) similar to the enzyme as prepared. Thus, Cl- binding prevents a rapi
d reaction of NO with the oxidized binuclear center. EPR experiments show n
o new signals within 15 a after addition of NO to the enzyme as prepared. A
mperometric measurements show that the pulsed NO-reactive enzyme reacts wit
h high affinity and a stoichiometry of 1 NO/aa(3), whereas the enzyme as pr
epared reacts to a very small extent (<20%). In both cases, the reactivity
is abolished by pre-incubation with cyanide. These experiments suggest that
the effect of "pulsing" the enzyme, which leads to enhanced NO reactivity,
arises from removing Cl- bound at the oxidized cytochrome a(3)-Cu-B site.