Abstract
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 (lambdamax = 424 nm) reacts with NO at k = 2.2 x 10(5) M-1 s-1 at 20 degreesC and is stable for hours unless Cl- is added, in which case it decays slowly (t1/2 approximately 70 min) to an unreactive state (lambdamax = 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 s 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/aa3, 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 a3-CuB site.
Highlights
The explosion of interest in the biological effects of nitric oxide (NO) has led to the discovery that this remarkable gas is a reversible inhibitor of COX1 [1,2,3] and may play an important role in the regulation of cellular respiration in vivo [4]
When NO is mixed with oxidized COX, a large absorbance change is seen with the pulsed enzyme, whereas with the enzyme as prepared only a small signal is detected (Fig. 1)
The reaction corresponds to the reduction of 50 – 60% of cytochrome a, with no spectral changes assigned to cytochrome a3 showing, in agreement with earlier work [11,12,13], that the reaction with NO is only observed when the enzyme is subjected to a “pulsing” protocol
Summary
The explosion of interest in the biological effects of NO has led to the discovery that this remarkable gas is a reversible inhibitor of COX1 [1,2,3] and may play an important role in the regulation of cellular respiration in vivo [4]. Kinetic studies aimed at understanding the mechanism of this inhibition [3, 5] or those involving NO as a trapping ligand for reduced cytochrome a3 [6] failed to reveal a fast reaction between NO and the oxidized enzyme as prepared. This is in agreement with previous studies [7,8,9,10] which showed that a reaction with oxidized COX was only observed following prolonged. In addition it has been suggested that when the enzyme undergoes a cycle of reduction and reoxidation (i.e. is pulsed), ClϪ dissociates [15]
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