Formation of the "Peroxy" Intermediate in Cytochrome c Oxidase Is Associated with Internal Proton/Hydrogen Transfer

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• 作者：Martin Karpefors ; Pia Ä ; delroth ; Andreas Namslauer ; Yuejun Zhen ; and Peter Brzezinski
• 刊名：Biochemistry
• 出版年：2000
• 出版时间：November 28, 2000
• 年：2000
• 卷：39
• 期：47
• 页码：14664 - 14669
• 全文大小：70K
• 年卷期：v.39,no.47(November 28, 2000)
• ISSN：1520-4995

文摘

When dioxygen is reduced to water by cytochrome c oxidase a sequence of oxygen intermediatesare formed at the reaction site. One of these intermediates is called the "peroxy" (P) intermediate. It canbe formed by reacting the two-electron reduced (mixed-valence) cytochrome c oxidase with dioxygen(called P_m), but it is also formed transiently during the reaction of the fully reduced enzyme with oxygen(called P_r). In recent years, evidence has accumulated to suggest that the O-O bond is cleaved in the Pintermediate and that the heme a₃ iron is in the oxo-ferryl state. In this study, we have investigated thekinetic and thermodynamic parameters for formation of P_m and P_r, respectively, in the Rhodobactersphaeroides enzyme. The rate constants and activation energies for the formation of the P_r and P_mintermediates were 1.4 × 10⁴ s^-1 (~20 kJ/mol) and 3 × 10³ s^-1 (~24 kJ/mol), respectively. The formationrates of both P intermediates were independent of pH in the range 6.5-9, and there was no proton uptakefrom solution during P formation. Nevertheless, formation of both P_m and P_r were slowed by a factor of1.4-1.9 in D₂O, which suggests that transfer of an internal proton or hydrogen atom is involved in therate-limiting step of P formation. We discuss the origin of the difference in the formation rates of the P_mand P_r intermediates, the formation mechanisms of P_m/P_r, and the involvement of these intermediates inproton pumping.