Interflavin Oxidation-Reduction Reactions between Pig Kidney General Acyl-CoA Dehydrogenase and Electron-Transferring Flavoprotein

Robert J. Gorelick, Lawrence M. Schopfer, David P. Ballou, Vincent Massey, Colin Thorpe

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Abstract

The mechanism of interflavin electron transfer between pig kidney general acyl-CoA dehydrogenase (GAD) and its physiological acceptor, electron-transferring flavoprotein (ETF), has been studied by static and stopped-flow absorbance and fluorescence measurements. At 3 °C, pH 7.6, reoxidation of the dehydrogenase (stoichiometrically reduced by octanoyl-CoA) by ETF is multiphasic, consisting of two rapid phases (t1/2 of about 20 and 50 ms), a slower phase half-complete in about 1 s, and a final reaction with a half-time of 20 s. Only the two most rapid phases are significant in turnover. This complicated reaction course was dissected by examining the rates of plausible individual steps, e.g., GAD2e-P + ETFle, GADle-P + ETF0X, and GADle-P + ETFle (where P represents the product, octenoyl-CoA, and the subscripts indicate the redox state of the flavin). Rapid reaction and static fluorescence measurements, in all cases, showed that the final equilibrium mixture included appreciable levels of oxidized ETF. This was confirmed by measuring the reverse reactions, e.g., ETFle + GAD0X-P, ETFle + GAD1e-P, and ETF2e + GAD0X-P. These data support the following overall scheme for the reaction of GAD2e-P with ETF0X: The first and second phases correspond to reoxidation of GAD2e-P in two successive one-electron steps requiring two molecules of ETF0X. This results in a rapid rise in absorbance at 370 nm where the red anionic radicals of both product-complexed dehydrogenase and ETF absorb strongly. The slower decline in 370-nm absorbance reflects further reduction of ETFle by one- and two-electron-reduced forms of the product-complexed enzyme. In accord with the proposed scheme, the disproportionation of ETF semiquinone is catalyzed by the dehydrogenase in the presence of octenoyl-CoA, and this disproportionation reaction contributes to the attainment of the final equilibrium in the slower phases. In the absence of bound product, the reduction of ETF0X by GAD2e follows a different course, proceeding much more slowly to completion. Initial one-electron transfer generates the blue dehydrogenase semiquinone (t1/2 = 600 ms at 3 °C) with concomitant formation of ETF red radical. Further reduction of ETFle proceeds very slowly (f1/2 = 60 s), with concomitant reoxidation of the dehydrogenase radical. These data identify the important role played by acyl-CoA product in modulation of the thermodynamic and kinetic behavior of the dehydrogenase during the reoxidation of substrate-reduced enzyme by electron-transferring flavoprotein.

Original languageEnglish (US)
Pages (from-to)6830-6839
Number of pages10
JournalBiochemistry
Volume24
Issue number24
DOIs
StatePublished - Nov 1 1985

ASJC Scopus subject areas

  • Biochemistry

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