Resonance Raman/absorption characterization of the oxo intermediates of cytochrome c oxidase generated in its reaction with hydrogen peroxide: pH and H2O2 concentration dependence.
Resonance Raman/absorption characterization of the oxo intermediates of cytochrome c oxidase generated in its reaction with hydrogen peroxide: pH and H2O2 concentration dependence.
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细胞色素 c 氧化酶与过氧化氢反应产生的氧化中间体的共振拉曼/吸收表征：pH 和 H2O2 浓度依赖性。
DOI：
10.1021/bi952096t
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发表时间：
1996
期刊：
影响因子：
2.9
通讯作者：
T. Kitagawa
中科院分区：
文献类型：
--
作者：
D. Proshlyakov;T. Ogura;K. Shinzawa;S. Yoshikawa;T. Kitagawa
利用先前报道的高性能拉曼/吸收同时测定技术研究了pH和H2O2浓度对细胞色素c氧化酶(CcO)与H2O2反应的影响(Proshlyakov等，1996)。该反应生成两种中间体，分别为 607 nm 和 580 nm 形式，我们发现它们显示出与 CcO 还原 O2 中的中间体相同的氧同位素敏感 RR 带。在单周转条件下获得的瞬态吸收光谱中，607 nm 形式出现为主要中间体，随后出现 580 nm 和静止形式，表明 H2O2 作为静止酶的氧化剂，但在过氧化物循环中作为 607 nm 和 580 nm 形式的还原剂。 607 nm 处的吸收上升速率对 H2O/D2O 交换不敏感，但 D2O 中的衰减速度明显慢于 H2O 中。在微循环系统中，通过持续供应H2O2，各中间体在稳态条件下保持在恒定水平。在 7.4 和 10.0 之间的 pH 范围内，607 nm 形式的数量在较高 pH 值和较高 H2O2 浓度下减少。在 804/769 cm-1 处观察到 H2(16)O2/H2(18)O2 衍生物的 607 nm 形式的氧代血红素的 Fe=O 拉伸 (VFe=O) 频率在此 pH 范围内没有改变，甚至在 pH 10.0 时也表现出 D2O/H2O 位移。这表明在该 pH 范围内，铁结合氧与远端残基形成氢键键合。当580 nm形式在H2O2的非饱和水平下占主导地位时，在中性pH下在785/750 cm-1和355/340 cm-1处观察到另外两个氧同位素敏感拉曼谱带，但前者在pH 8.5以上消失，后者在pH 9.0以上消失，吸收光谱没有显着变化，表明以580 nm形式存在两个不同的物种。然而，在 H2O2 饱和浓度下，这些拉曼谱带在 pH 7.4 和 10.0 之间没有变化。相反，在没有过量过氧化物的情况下，尽管 580 nm 形式占主导地位，但没有观察到氧同位素敏感 RR 带。这些拉曼带的消失表明氧代血红素和大量水之间发生了氧交换，其速率超过了在碱性 pH 值和/或低 H2O2 浓度下 580 nm 形式的形成速率。这种氧交换在 607 nm 形式下不会发生。在产生特定稳态的相同实验条件下，H2O 与 D2O 的交换导致 580 nm 形式的显着减少，并伴随 607 nm 形式的增加。根据 H2O 和 D2O 之间 607 nm 形式的衰减率差异，这一点得到了令人满意的解释。因此，607 nm 形式还原为 580 nm 形式可能是 O2 还原中氧化还原连接质子泵浦的关键步骤。
Effects of pH and H2O2 concentration on the reaction of cytochrome c oxidase (CcO) with H2O2 were studied with the high-performance Raman/absorption simultaneous determination technique reported previously (Proshlyakov et al., 1996). This reaction generates two intermediates called 607- and 580-nm forms, and we found that they show the same oxygen-isotope-sensitive RR bands as those of the intermediates in O2 reduction by CcO. In transient absorption spectra obtained under single turnover conditions, the 607-nm form appeared as the primary intermediate and subsequently the 580-nm and resting forms, suggesting that H2O2 serves as an oxidant for the resting enzyme but as a reductant for both the 607- and 580-nm forms in the peroxide cycle. The rise rate of absorption at 607 nm was insensitive to the H2O/D2O exchange, but the decay was significantly slower in D2O than in H2O. With the microcirculating system, each intermediate was maintained at a constant level under steady-state conditions by supplying H2O2 continuously. In the pH range between 7.4 and 10.0, the population of the 607-nm form decreased at higher pH and at higher concentrations of H2O2. The Fe=O stretching (VFe=O) frequencies of the oxo heme of the 607-nm form, observed at 804/769 cm-1 for their H2(16)O2/H2(18)O2 derivatives, were unaltered in this pH range and exhibited a D2O/H2O shift even at pH 10.0. This indicates that the iron-bound oxygen is hydrogen-bonded to a distal residue in this pH range. When the 580-nm form is dominant under the nonsaturating level of H2O2, two other oxygen-isotope-sensitive Raman bands have been observed at 785/750 cm-1 and 355/340 cm-1 at neutral pH, but the former disappeared above pH 8.5 and the latter above pH 9.0 without significant changes of absorption spectra, suggesting the presence of two separate species in the name of the 580-nm form. However, under the saturating concentration of H2O2, these Raman bands were unaltered between pH 7.4 and 10.0. In contrast, in the absence of excess peroxide, no oxygen-isotope-sensitive RR bands were observed despite dominance of the 580-nm form. The disappearance of these Raman bands demonstrates the occurrence of oxygen exchange between the oxo heme and bulk water, whose rate surpasses the formation rate of the 580-nm form at alkaline pH and/or at low H2O2 concentration. Such an oxygen exchange did not take place in the 607-nm form. Under the identical experimental conditions for generating a particular steady state, the exchange of H2O with D2O caused significant depopulation of the 580-nm form and concomitant increase of the 607-nm form. This was satisfactorily interpreted in terms of the difference in the decay rate of the 607-nm form between H2O and D2O. Thus, the reduction of the 607-nm form to the 580-nm form is likely to be a key step of the redox-linked proton pumping in the O2 reduction.