Kinetics of the spectral changes during reduction of the Na+-motive NADH:quinone oxidoreductase from Vibrio harveyi

被引:36
作者
Bogachev, AV
Bertsova, YV
Ruuge, EK
Wikström, M
Verkhovsky, MI [1 ]
机构
[1] Moscow MV Lomonosov State Univ, AN Belozersky Inst Physicochem Biol, Dept Bioenerget, Moscow 119899, Russia
[2] Cardiol Res Ctr, Res Inst Expt Cardiol, Moscow 121552, Russia
[3] Univ Helsinki, Inst Biotechnol, Helsinki Bioenerget Grp, FIN-00014 Helsinki, Finland
来源
BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS | 2002年 / 1556卷 / 2-3期
基金
芬兰科学院; 俄罗斯基础研究基金会;
关键词
Na+-NADH : quinone oxidoreductase; flavin cofactor; Na+-pump; respiratory enzyme; bacteria; Vibrio harveyi;
D O I
10.1016/S0005-2728(02)00342-0
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Two radical signals with different line widths are seen in the Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) from Vibrio harveyi by EPR spectroscopy. The first radical is observed in the oxidized enzyme, and is assigned as a neutral flavosemiquinone. The second radical is observed in the reduced enzyme and is assigned to be the anionic form of flavosemiquinone. The time course of Na+-NQR reduction by NADH, as monitored by stopped-flow optical spectroscopy, shows three distinct phases, the spectra of which suggest that they correspond to the reduction of three different flavin species. The first phase is fast both in the presence and absence of sodium, and is assigned to reduction of FAD to FADH(2) at the NADH dehydrogenating site. The rates of the other two phases are strongly dependent on sodium concentration, and these phases are attributed to reduction of two covalently bound FMN's. Combination of the optical and EPR data suggests that a neutral FMN flavosemiquinone preexists in the oxidized enzyme, and that it is reduced to the fully reduced flavin by NADH. The other FMN moiety is initially oxidized, and is reduced to the anionic flavosemiquinone. One-electron transitions of two discrete flavin species are thus assigned as sodium-dependent steps in the catalytic cycle of Na+-NQR. (C) 2002 Elsevier Science B.V. All rights reserved.
引用
收藏
页码:113 / 120
页数:8
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