→H+/2e stoichiometry in NADH‐quinone reductase reactions catalyzed by bovine heart submitochondrial particles

FEBS Letters

Vinogradov, A.D.¹  Galkin, A.S.¹  Grivennikova, V.G.¹ 

[1] Department of Biochemistry, School of Biology, Moscow State University, Moscow 119899, Russian Federation

关键词: NADH-ubiquinone oxidoreductase;    Complex I;    Energy transduction;    Respiratory chain;    Bovine heart mitochondrion;    PhR and NR;    pH indicators phenol red and neutral red;    respectively;    FCCP;    carbonyl cyanide p-(trifluoromethoxy) phenyl-hydrazone;    BSA;    bovine serum albumin;    DB;    2;    3-dimethoxy-5-methyl-6-decylbenzoquinone;    Qn;    homologues of ubiquinone having n isoprenoid units in position 6 of the quinone ring;    NEM;    N-ethylmaleimide;   

DOI  :  10.1016/S0014-5793(99)00575-X

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Tightly coupled bovine heart submitochondrial particles treated to activate complex I and to block ubiquinol oxidation were capable of rapid uncoupler-sensitive inside-directed proton translocation when a limited amount of NADH was oxidized by the exogenous ubiquinone homologue Q₁. External alkalization, internal acidification and NADH oxidation were followed by the rapidly responding (t _1/2≤1 s) spectrophotometric technique. Quantitation of the initial rates of NADH oxidation and external H⁺ decrease resulted in a stoichiometric ratio of 4 H⁺ vectorially translocated per 1 NADH oxidized at pH 8.0. ADP-ribose, a competitive inhibitor of the NADH binding site decreased the rates of proton translocation and NADH oxidation without affecting →H⁺/2e stoichiometry. Rotenone, piericidin and thermal deactivation of complex I completely prevented NADH-induced proton translocation in the NADH-endogenous ubiquinone reductase reaction. NADH-exogenous Q₁ reductase activity was only partially prevented by rotenone. The residual rotenone- (or piericidin-) insensitive NADH-exogenous Q₁ reductase activity was found to be coupled with vectorial uncoupler-sensitive proton translocation showing the same →H⁺/2e stoichiometry of 4. It is concluded that the transfer of two electrons from NADH to the Q₁-reactive intermediate located before the rotenone-sensitive step is coupled with translocation of 4 H⁺.

【 授权许可】

Unknown