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→H + /2e stoichiometry in NADH‐quinone reductase reactions catalyzed by bovine heart submitochondrial particles
Author(s) -
Galkin A.S.,
Grivennikova V.G.,
Vinogradov A.D.
Publication year - 1999
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(99)00575-x
Subject(s) - submitochondrial particle , rotenone , chemistry , reductase , stoichiometry , nadh dehydrogenase , electron transport chain , biochemistry , photochemistry , enzyme , mitochondrion , protein subunit , organic chemistry , gene
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 Q 1 . 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 Q 1 reductase activity was only partially prevented by rotenone. The residual rotenone‐ (or piericidin‐) insensitive NADH‐exogenous Q 1 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 Q 1 ‐reactive intermediate located before the rotenone‐sensitive step is coupled with translocation of 4 H + .