Giuffrè A, Stubauer G, Brunori M, Sarti P, Torres J, Wilson M T
Department of Biochemical Sciences and Consiglio Nazionale delle Ricerche Center of Molecular Biology, University of Rome "La Sapienza", I-00185 Rome, Italy.
J Biol Chem. 1998 Dec 4;273(49):32475-8. doi: 10.1074/jbc.273.49.32475.
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.
采用停流法、安培法和电子顺磁共振（EPR）技术，研究了一氧化氮（NO）与氧化态快速细胞色素c氧化酶的反应，所用酶为制备好的或经过“脉冲处理”后的。观察到经脉冲处理的细胞色素a迅速还原，而制备好的酶则未出现这种情况。反应物种（λmax = 424 nm）在20℃下以k = 2.2×10⁵ M⁻¹ s⁻¹的速率与NO反应，并且在数小时内保持稳定，除非加入Cl⁻，在这种情况下它会缓慢衰减（t1/2约为70分钟）至与制备好的酶类似的无反应状态（λmax = 423 nm）。因此，Cl⁻的结合可防止NO与氧化态双核中心快速反应。EPR实验表明，向制备好的酶中加入NO后15秒内未出现新信号。安培测量表明，经脉冲处理的NO反应性酶以高亲和力和1个NO/aa3的化学计量比发生反应，而制备好的酶反应程度非常小（<20%）。在两种情况下，预先用氰化物孵育均可消除反应活性。这些实验表明，对酶进行“脉冲处理”导致NO反应性增强的效应，是由于去除了结合在氧化态细胞色素a3-CuB位点上的Cl⁻。
