Mechanism of Redox-linked Proton Pumping in Terminal Enzyme of Cellular Respiration Studied by Pulse Radiolysis

来自大肠杆菌的细胞色素b型泛喹酚氧化酶属于血红素-铜末端氧化酶，是有氧呼吸链上氧化还原驱动的质子泵。为了了解质子泵的分子机制，我们对发生氧还原和质子转移的亚基1进行了定点突变。我们应用了脉冲辐解技术，这是研究单电子转移过程的有效方法之一，对缺乏CuB中心或具有D-通道或K-通道缺陷的亚基1突变体进行质子转移。在N-甲基烟酰胺存在下对野生型酶进行脉冲辐解，观察到在Q_H位产生440 nm宽峰的泛半喹酮阴离子自由基，随后电子转移到血红素b和o，一级电子转移为1.5×10~(-3)S^~(-1)~(-1)。在His33ala中，观察到了血红素的两相还原，其速率常数分别为1.1x10^5 S^-1&gt；和8.9x10^2 S^-1&gt；，表明在血红素到血红素的电子转移中存在微扰。在D途径突变的酶(E286D、D135N)表现出与野生型酶相同的时间常数。在K途径变异体K362Q和Y288F中，从血红素b到o的分子内电子转移减少。这些结果表明，在从氧化到单电子还原态的转变过程中，质子通过K途径吸收。与野生型酶相比，K突变酶的Em值提高了125 mV。此外，随着分子内电子转移的减少，血红素b和o之间的氧化还原电位之差也增大。这表明电子转移是由质子吸收控制的。

Cytochrome bo-type ubiquinol oxidase from Escherichia coli belongs to the heme-copper terminal oxidase and serves as a redox-driven proton pump of the aerobic respiratory chain. To understand the molecular mechanism of proton pumping, we have been carrying out site-directed mutagenesis on subunit 1, where dioxygen reduction and proton translocation take place. We applied pulse radiolysis technique, one of the powerful methods for studying one-electron transfer processes, to subunit 1 mutants lacking the Cu_B center or having defects either in D-or K-channel for proton translocation. Upon pulse radiolysis of the wild-type enzyme in the presence of N-methyl nicotinamide as an electron mediator, we observed the generation of ubisemiquinone anion radical with a broad peak at 440 nm at the Q_H site and subsequent electron transfer to hemes b and o with a first-order of 1.5 x 10^3 s^<-1>. In His333ala, a biphasic reduction of the hemes with the rate constants of 1.1 x 10^5 s^<-1> and 8.9 x 10^2 s^<-1> was observed, indicating that the perturbation in heme-to heme electron transfer. The enzymes variants mutated in the D pathway of proton transfer (E286D, D135N) show the same time constants as the wild-type enzyme. In the K pathway variant K362Q and Y288F, on the other hand, the intramolecular electron transfer from heme b to o were decreased. These results suggest that uptake of a proton through the K pathway during the transition from the oxidized to the one-electron reduced state. The E_m values for the K mutant enzymes were increased by 125 mV as the wild-type enzyme. Moreover, the difference in the redox potentials between heme b and o increased with the decrease of the intramolecular electron transfer. This suggests that electron transfer is controlled by the proton uptake.