Giuffrè A, Sarti P, D'Itri E, Buse G, Soulimane T, Brunori M
Department of Biochemical Sciences and Consiglio Nazionale delle Ricerche Center of Molecular Biology, University of Rome "La Sapienza," 00185 Rome, Italy.
J Biol Chem. 1996 Dec 27;271(52):33404-8. doi: 10.1074/jbc.271.52.33404.
The mechanism of inhibition of cytochrome (cyt) c oxidase by nitric oxide (NO) has been investigated by stopped flow transient spectroscopy and singular value decomposition analysis. Following the time course of cyt c oxidation at different O2/NO ratios, we observed that the onset of inhibition: (i) is fast and at a high NO concentration is complete during the first turnover; (ii) is sensitive to the O2/NO ratio; and (iii) is independent of incubation time of the oxidized enzyme with NO. Analysis of the reaction kinetics and computer simulations support the conclusion that inhibition occurs via binding of NO to a turnover intermediate with a partially reduced cyt a3-CuB binuclear center. The inhibited enzyme has the optical spectrum typical of NO bound to reduced cyt a3. Reversal of inhibition in the presence of O2 does not involve a direct reaction of O2 with NO while bound at the binuclear center, since recovery of activity occurs at the rate of NO dissociation (k = 0.13 s-1), as determined in the absence of O2 using hemoglobin as a NO scavenger. We propose that removal of NO from the medium is associated with reactivation of the enzyme via a relatively fast thermal dissociation of NO from the reduced cyt a3-CuB center.
通过停流瞬态光谱和奇异值分解分析研究了一氧化氮（NO）对细胞色素（cyt）c氧化酶的抑制机制。在不同O₂/NO比例下跟踪cyt c氧化的时间进程，我们观察到抑制的起始：（i）很快，在高NO浓度下，第一次周转期间抑制就完成了；（ii）对O₂/NO比例敏感；（iii）与氧化酶与NO的孵育时间无关。反应动力学分析和计算机模拟支持这样的结论，即抑制是通过NO与具有部分还原的cyt a₃-CuB双核中心的周转中间体结合而发生的。被抑制的酶具有NO与还原型cyt a₃结合的典型光谱。在O₂存在下抑制的逆转并不涉及O₂与结合在双核中心的NO的直接反应，因为活性的恢复以NO解离速率（k = 0.13 s⁻¹）发生，这是在不存在O₂的情况下使用血红蛋白作为NO清除剂测定的。我们提出，通过从还原型cyt a₃-CuB中心相对快速的热解离将NO从培养基中去除与酶的重新激活有关。
