Zaslavsky D, Sadoski R C, Wang K, Durham B, Gennis R B, Millett F
School of Chemical Sciences, University of Illinois, Urbana 61801, USA.
Biochemistry. 1998 Oct 20;37(42):14910-6. doi: 10.1021/bi981490z.
The final step of the catalytic cycle of cytochrome oxidase, the reduction of oxyferryl heme a3 in compound F, was investigated using a binuclear polypyridine ruthenium complex (Ru2C) as a photoactive reducing agent. The net charge of +4 on Ru2C allows it to bind electrostatically near CuA in subunit II of cytochrome oxidase. Photoexcitation of Ru2C with a laser flash results in formation of a metal-to-ligand charge-transfer excited state, Ru2C, which rapidly transfers an electron to CuA of cytochrome oxidase from either beef heart or Rhodobacter sphaeroides. This is followed by reversible electron transfer from CuA to heme a with forward and reverse rate constants of k1 = 9.3 x 10(4) s-1 and k-1 = 1.7 x 10(4) s-1 for R. sphaeroides cytochrome oxidase in the resting state. Compound F was prepared by treating the resting enzyme with excess hydrogen peroxide. The value of the rate constant k1 is the same in compound F where heme a3 is in the oxyferryl form as in the resting enzyme where heme a3 is ferric. Reduction of heme a in compound F is followed by electron transfer from heme a to oxyferryl heme a3 with a rate constant of 700 s-1, as indicated by transients at 605 and 580 nm. No delay between heme a reoxidation and oxyferryl heme a3 reduction is observed, showing that no electron-transfer intermediates, such as reduced CuB, accumulate in this process. The rate constant for electron transfer from heme a to oxyferryl heme a3 was measured in beef cytochrome oxidase from pH 7.0 to pH 9.5, and found to decrease upon titration of a group with a pKa of 9.0. The rate constant is slower in D2O than in H2O by a factor of 4.3, indicating that the electron-transfer reaction is rate-limited by a proton-transfer step. The pH dependence and deuterium isotope effect for reduction of isolated compound F are comparable to that observed during reaction of the reduced, CO-inhibited CcO with oxygen by the flow-flash technique. This result indicates that electron transfer from heme a to oxyferryl heme a3 is not controlled by conformational effects imposed by the initial redox state of the enzyme. The rate constant for electron transfer from heme a to oxyferryl heme a3 is the same in the R. sphaeroides K362M CcO mutant as in wild-type CcO, indicating that the K-channel is not involved in proton uptake during reduction of compound F.
使用双核多吡啶钌配合物（Ru2C）作为光活性还原剂，研究了细胞色素氧化酶催化循环的最后一步，即化合物F中氧合铁血红素a3的还原。Ru2C上的 +4 净电荷使其能够静电结合在细胞色素氧化酶亚基 II 中的 CuA 附近。用激光闪光对 Ru2C 进行光激发会导致形成金属 - 配体电荷转移激发态 Ru2C*，它会迅速将一个电子从牛肉心或球形红细菌的细胞色素氧化酶的 CuA 转移过去。接下来是从 CuA 到血红素 a 的可逆电子转移，对于处于静止状态的球形红细菌细胞色素氧化酶，正向和反向速率常数分别为 k1 = 9.3×10⁴ s⁻¹ 和 k⁻¹ = 1.7×10⁴ s⁻¹。通过用过量过氧化氢处理静止酶来制备化合物F。在化合物F中，血红素a3处于氧合铁形式时的速率常数k1与血红素a3为三价铁的静止酶中的相同。化合物F中血红素a的还原之后是从血红素a到氧合铁血红素a3的电子转移，速率常数为700 s⁻¹，这由605和580 nm处的瞬态信号表明。未观察到血红素a再氧化和氧合铁血红素a3还原之间的延迟，这表明在此过程中没有电子转移中间体（如还原的CuB）积累。在pH 7.0至pH 9.5范围内测量了牛肉细胞色素氧化酶中从血红素a到氧合铁血红素a3的电子转移速率常数，发现用pKa为9.0的基团进行滴定后该常数会降低。在D₂O中速率常数比在H₂O中慢4.3倍，这表明电子转移反应受质子转移步骤的速率限制。分离的化合物F还原的pH依赖性和氘同位素效应与通过流动闪光技术观察到的还原的、CO抑制的细胞色素c氧化酶与氧气反应期间的情况相当。该结果表明，从血红素a到氧合铁血红素a3的电子转移不受酶的初始氧化还原状态所施加的构象效应的控制。在球形红细菌K362M细胞色素c氧化酶突变体中，从血红素a到氧合铁血红素a3的电子转移速率常数与野生型细胞色素c氧化酶中的相同，这表明在化合物F还原过程中K通道不参与质子摄取。
