Han S, Takahashi S, Rousseau D L
Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
J Biol Chem. 2000 Jan 21;275(3):1910-9. doi: 10.1074/jbc.275.3.1910.
Cytochrome c oxidase, the terminal enzyme in the electron transfer chain, catalyzes the reduction of oxygen to water in a multiple step process by utilizing four electrons from cytochrome c. To study the reaction mechanism, the resonance Raman spectra of the intermediate states were measured during single turnover of the enzyme after catalytic initiation by photolysis of CO from the fully reduced CO-bound enzyme. By measuring the change in intensity of lines associated with heme a, the electron transfer steps were determined and found to be biphasic with apparent rate constants of approximately 40 x 10(3) s(-1) and approximately 1 x 10(3) s(-1). The time dependence for the oxidation of heme a and for the measured formation and decay of the oxy, the ferryl ("F"), and the hydroxy intermediates could be simulated by a simple reaction scheme. In this scheme, the presence of the "peroxy" ("P") intermediate does not build up a sufficient population to be detected because its decay rate is too fast in buffered H(2)O at neutral pH. A comparison of the change in the spin equilibrium with the formation of the hydroxy intermediate demonstrates that this intermediate is high spin. We also confirm the presence of an oxygen isotope-sensitive line at 355 cm(-1), detectable in the spectrum from 130 to 980 micros, coincident with the presence of the F intermediate.
细胞色素c氧化酶是电子传递链中的末端酶，通过利用来自细胞色素c的四个电子，在一个多步骤过程中催化氧气还原为水。为了研究反应机制，在通过光解完全还原的一氧化碳结合酶中的一氧化碳引发催化后，在酶的单次周转过程中测量了中间态的共振拉曼光谱。通过测量与血红素a相关的谱线强度变化，确定了电子传递步骤，发现其为双相的，表观速率常数约为40×10³ s⁻¹和约1×10³ s⁻¹。血红素a氧化的时间依赖性以及所测量的氧合、高铁（“F”）和羟基中间体的形成与衰减可以通过一个简单的反应方案进行模拟。在该方案中，“过氧”（“P”）中间体的存在不会积累到足以被检测到的数量，因为在中性pH的缓冲H₂O中其衰减速率太快。自旋平衡变化与羟基中间体形成的比较表明，该中间体是高自旋的。我们还证实了在355 cm⁻¹处存在一条对氧同位素敏感的谱线，在130至980微秒的光谱中可检测到，与F中间体的存在一致。
