Chloride bound to oxidized cytochrome c oxidase controls the reaction with nitric oxide

The reaction of nitric oxide (NO) with oxidized fast cytochrome c oxidase was 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 degrees C and is stable for hours unless Cl- is added, in which ease it decays slowly (t(1/2) similar to 70 min) to an unreactive state (lambda(max) = 423 nm) similar to the enzyme as prepared. Thus, Cl- binding prevents a rapid reaction of NO with the oxidized binuclear center. EPR experiments show no new signals within 15 a after addition of NO to the enzyme as prepared. Amperometric measurements show that the pulsed NO-reactive enzyme reacts with high affinity and a stoichiometry of 1 NO/aa(3), whereas the enzyme as prepared 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.