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

脉冲放射分解研究细胞呼吸终末酶氧化还原连接质子泵浦机制

• 批准号: 14380318
• 负责人: KOBAYASHI Kazuo
• 金额: $ 8.7万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14380318/
• 关键词: pulse radiolysis; cytochrome oxidase; proton pump; electron transfer; oxidation-reduction potential; channel; heme; 末端酸化酵素; プロトン輸送; プロトンチャネル; ユビキノール; 変異体

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.

大肠杆菌细胞色素bo型泛醇氧化酶属于血红素-铜末端氧化酶，是有氧呼吸链的氧化还原驱动质子泵。为了了解质子泵的分子机制，我们对亚基1进行了定点突变，其中发生了分子氧还原和质子转运。我们应用脉冲辐解技术研究了单电子转移过程中的一种强有力的方法，对缺乏Cu_B中心或质子转运的D-或K-通道缺陷的亚基1突变体进行了研究。在N-甲基烟酰胺作为电子介质存在下，对野生型酶进行脉冲辐解后，我们观察到在Q_H位点产生了泛半醌阴离子自由基，在440 nm处有一个宽峰，随后电子转移到血红素B和o上，一级反应速率为1.5 x 10^3 s^2<-1>。在His 333 ala中，观察到血红素的双相还原，速率常数为1.1 x 10^5 s^<-1>和8.9 x 10^2 s^<-1>，表明血红素-血红素电子转移中的扰动。在质子转移的D途径中突变的酶变体（E286 D，D135 N）显示与野生型酶相同的时间常数。另一方面，在K途径变体K362 Q和Y288 F中，从血红素B到o的分子内电子转移减少。这些结果表明，在从氧化态到单电子还原态的过渡期间，通过K途径吸收质子。K突变酶的E_m值比野生型酶增加了125 mV。血红素B和血红素O的氧化还原电位差随分子内电子转移的减少而增大。这表明电子转移是由质子吸收控制的。

项目成果

K S.matsuura, S.Yoshioka, K.Takahashi, T.Ishimori, T.Mogi, H.Hori, I.Morishima: "Dioxygen reduction by bo-type quinol oxidase from Escherichia coli by submillisecond-resolved freeze quench EPR spectroscopy"Biochemistry. 43. (2004)

K S.matsuura、S.Yoshioka、K.Takahashi、T.Ishimori、T.Mogi、H.Hori、I.Morishima：“通过亚毫秒分辨冷冻淬灭 EPR 光谱法，通过大肠杆菌中的 bo 型对苯二酚氧化酶还原双氧”

Koji Matsuura, Shiro Yoshioka, Satoshi Takahashi, Koichiro Ishimori, Tatushi Mogi, Isao Morishima: "The observation of the dioxygen activation of cytochrome bo from Escherichia coli by submillisecond-resolved freeze quench EPR spectroscopy"J.Inorg.Biochem

Koji Matsuura、Shiro Yoshioka、Satoshi Takahashi、Koichiro Ishimori、Tatushi Mogi、Isao Morishima：“通过亚毫秒分辨冷冻淬灭 EPR 光谱观察大肠杆菌细胞色素 bo 的双氧活化”J.Inorg.Biochem

S.T.Grimaldi, N.Ostermann, N.Weiden, T.Mogi, H.Miyoshi, B.Ludwig, H.Michel, T.F.Prisner, F.MacMillan: "Asymmetric binding of the high-affinity Q_H ubisemiquinone in quinol oxidase (bo_3) from Escherichia coli studied by multifrequency electron paramagneti

S.T.Grimaldi、N.Ostermann、N.Weiden、T.Mogi、H.Miyoshi、B.Ludwig、H.Michel、T.F.Prisner、F.MacMillan：“高亲和力 Q_H 泛半醌在醌醇氧化酶 (bo_3) 中的不对称结合

Kazuo Kobayashi, Seiichi Tagawa: "Direct Observation of Guanine Radical Cation Deprotonation in Duplex DNA Using Pulse Radiolysis"J.Amer.Chem.Soc.. 125. 10213-10218 (2003)

Kazuo Kobayashi、Seiichi Takawa：“使用脉冲放射分解直接观察双链 DNA 中鸟嘌呤自由基阳离子去质子化”J.Amer.Chem.Soc.. 125. 10213-10218 (2003)

Deligeer, R.Fukunaga, K.Kataoka, K.Yamaguchi, K.Kobayashi, S.Tagawa, S.Suzuki: "Spectroscopic and functional characterization of CuOcontaining nitrite reductase from Hyphomicrobium Denitrificans A3151"J.Inorg.Biochem.. 91. 132-138 (2002)

Deligeer、R.Fukunaga、K.Kataoka、K.Yamaguchi、K.Kobayashi、S.Takawa、S.Suzuki：“来自脱氮菌 A3151 的含 CuO 亚硝酸还原酶的光谱和功能表征”J.Inorg.Biochem.. 91. 132