Pecoraro C, Gennis R B, Vygodina T V, Konstantinov A A
Department of Biochemistry, University of Illinois, Urbana 61801, USA.
Biochemistry. 2001 Aug 14;40(32):9695-708. doi: 10.1021/bi010115v.
The reaction of cytochrome c oxidase (COX) from Rhodobacter sphaeroides with hydrogen peroxide has been studied at alkaline (pH 8.5) and acidic (pH 6.5) conditions with the aid of a stopped-flow apparatus. Absorption changes in the entire 350-800 nm spectral range were monitored and analyzed by a global fitting procedure. The reaction can be described by the sequential formation of two intermediates analogous to compounds I and II of peroxidases: oxidized COX + H2O2 --> intermediate I --> intermediate II. At pH as high as 8.5, intermediate I appears to be a mixture of at least two species characterized by absorption bands at approximately 607 nm (P607) and approximately 580 nm (F-I580) that rise synchronously. At acidic pH (6.5), intermediate I is represented mainly by a component with an alpha-peak around 575 nm (F-I575) that is probably equivalent to the so-called F* species observed with the bovine COX. The data are consistent with a pH-dependent reaction branching at the step of intermediate I formation. To get further insight into the mechanism of the pH-dependence, the peroxide reaction was studied using two mutants of the R. sphaeroides oxidase, K362M and D132N, that block, respectively, the proton-conducting K- and D-channels. The D132N mutation does not affect significantly the Ox --> intermediate I step of the peroxide reaction. In contrast, K362M replacement exerts a dramatic effect, eliminating the pH-dependence of intermediate I formation. The data obtained allow us to propose that formation of the acidic form of intermediate I (F-I575, F*) requires protonation of some group at/near the binuclear site that follows or is concerted with peroxide binding. The protonation involves specifically the K-channel. Presumably, a proton vacancy can be generated in the site as a consequence of the proton-assisted heterolytic scission of the O-O bond of the bound peroxide. The results are consistent with a proposal [Vygodina, T. V., Pecoraro, C., Mitchell, D., Gennis, R., and Konstantinov, A. A. (1998) Biochemistry 37, 3053-3061] that the K-channel may be involved in the delivery of the first four protons in the catalytic cycle (starting from reduction of the oxidized form) including proton uptake coupled to reduction of the binuclear site and transfer of protons driven by cleavage of the dioxygen O-O bond in the binculear site. Once peroxide intermediate I has been formed, generation of a strong oxene ligand at the heme a3 iron triggers a transition of the enzyme to the "peroxidase conformation" in which the K-channel is closed and the binuclear site becomes protonically disconnected from the bulk aqueous phase.
利用停流装置，研究了球形红杆菌细胞色素c氧化酶（COX）在碱性（pH 8.5）和酸性（pH 6.5）条件下与过氧化氢的反应。通过全局拟合程序监测并分析了整个350 - 800 nm光谱范围内的吸收变化。该反应可描述为依次形成两种类似于过氧化物酶化合物I和II的中间体：氧化型COX + H₂O₂→中间体I→中间体II。在高达pH 8.5时，中间体I似乎是至少两种物种的混合物，其特征在于在约607 nm（P607）和约580 nm（F - I580）处的吸收带同步上升。在酸性pH（6.5）下，中间体I主要由一个在约575 nm处有α峰的组分（F - I575）代表，该组分可能等同于在牛COX中观察到的所谓F物种。这些数据与中间体I形成步骤中pH依赖性反应分支一致。为了进一步深入了解pH依赖性的机制，使用球形红杆菌氧化酶的两个突变体K362M和D132N研究了过氧化物反应，这两个突变体分别阻断了质子传导的K通道和D通道。D132N突变对过氧化物反应的氧化型COX→中间体I步骤没有显著影响。相反，K362M替换产生了显著影响，消除了中间体I形成的pH依赖性。获得的数据使我们能够提出，中间体I酸性形式（F - I575，F）的形成需要双核位点处或其附近的某个基团质子化，该质子化跟随或与过氧化物结合协同发生。质子化具体涉及K通道。据推测，由于结合的过氧化物O - O键的质子辅助异裂，位点中可能产生质子空位。这些结果与一个提议[Vygodina, T. V., Pecoraro, C., Mitchell, D., Gennis, R., and Konstantinov, A. A. (1998) Biochemistry 37, 3053 - 3061]一致，即K通道可能参与催化循环中前四个质子的传递（从氧化型的还原开始），包括与双核位点还原偶联的质子摄取以及由双核位点中双氧O - O键断裂驱动的质子转移。一旦过氧化物中间体I形成，血红素a₃铁处强氧烯配体的产生会触发酶向“过氧化物酶构象”转变，其中K通道关闭，双核位点在质子方面与大量水相断开连接。
