Pig kidney general acyl-CoA dehydrogenase is rapidly, stoichiometrically, and irreversibly inactivated by the acetylenic thio ester 2-octynoyl coenzyme A (2-octynoyl-CoA). The inhibitor binds initially to the dehydrogenase with a 10-nm red shift and increased resolution of the flavin chromophore, followed by the generation of a charge-transfer complex between some form of the bound inhibitor and oxidized flavin (lambda max 800 nm; epsilon app = 4.5 mM-1 cm-1; k1 = 1.07 min-1, at pH 7.6, 25 degrees C). The rate of formation of the long wavelength band is increased markedly with increasing pH (pKapp = 7.9). This intermediate then decays with release of about 0.6 mol of CoASH at pH 7.6, yielding a final form with a spectrum typical of bound oxidized flavin. Both irreversible inactivation and covalent modification of the protein occur prior to the decay of the long wavelength species. The modified dehydrogenase is not reduced on prolonged anaerobic incubation with the substrate octanoyl-CoA. The inactive enzyme is unusually resistant to dithionite reduction but may be readily photoreduced via the blue semiquinone to the dihydroflavin form. This reduced enzyme is rapidly reoxidized by electron-transferring flavoprotein, the physiological electron acceptor of the dehydrogenase. General acyl-CoA dehydrogenase is also inactivated by 2-pentynoyl- and 2-pentadecynoyl-CoA with formation of an 800-nm band of lower intensity and by propiolyl-CoA, phenylpropiolyl-CoA, and 2-octynoylpantetheine without the appearance of detectable intermediate species. These data are compared with the behavior of acyl-CoA dehydrogenases toward mechanism-based inactivators carrying an acetylene function at C-3, e.g., 3-butynoyl-CoA.

Electron-transferring flavoprotein has been isolated from pig kidney by a simple procedure with a 7-fold higher yield over a previous method using pig liver. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, amino acid analysis, peptide mapping, and measurement of flavin content indicate that pig kidney electron-transferring flavoprotein contains nonidentical subunits (Mr 30 000 and 33 000) with one flavin adenine dinucleotide per dimer. These data contrast with reports that the liver protein is a dimer of identical subunits containing two flavin molecules. Dithionite and ferricyanide titrations indicate that flavin is the only redox-active moiety in pig kidney electron-transferring flavoprotein. Disproportionation of the anionic semiquinone is very slow, requiring about 10 h for half-completion. In contrast to results obtained with the liver protein, pig kidney electron-transferring flavoprotein does not bind crotonyl coenzyme A (crotonyl-CoA) significantly, and the semiquinone form is not reoxidized by crotonyl-CoA directly. These data do not support recent suggestions for a broader role of electron-transferring flavoprotein in beta oxidation.

Pig kidney general acyl-CoA dehydrogenases forms the blue neutral radical on dithionite or photochemical reduction (Thorpe, C., Matthews, R. G., & Williams, C. H. (1979) Biochemistry 18, 331-337] in accord with its classification as a flavoprotein dehydrogenase. However, dithionite reduction of the enzyme in the presence of crotonyl coenzyme A (crotonyl-CoA) or octenoyl-CoA generates the red radical anion as the predominant species at pH 7.6. Crotonyl-CoA binds preferentially to the red radical form, depressing the apparent pK by at least 2.5 pH units to a value of 7.3. Butyryl-, octanoyl-, and palmitoyl-CoA induce very similar spectral changes to those induced by enoyl-CoA derivatives when added anaerobically to the blue semiquinone enzyme. In contrast, the competitive inhibitors acetoacetyl-CoA and heptadecyl-SCoA do not markedly perturb the spectrum of the neutral flavosemiquinone species. The stability of the enzyme radical complexes with either crotonyl- or octanoyl-CoA suggests that there is not effective intraflavin transfer of reducing equivalents between subunits. Perturbation of the spectrum of the one-electron-reduced enzyme by ligands may complicate interpretation of the reaction enzyme by ligands may complicate interpretation of the reaction between substrate complexes of the general acyl-CoA dehydrogenase and electron-transferring flavoprotein.

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The flavoprotein pig kidney general acyl-CoA dehydrogenase contains a single catalytically essential methionine residue/FAD which reacts with iodoacetate at pH 6.6. S-Carboxymethylation of this residue generates an inactive enzyme derivative which retains FAD and the tetrameric structure of the native protein. The derivative binds actanoyl-CoA and palmityol-CoA with concomitant perturbation of the flavin chromophore, but the characterisitic spectrum of the reduced enzyme-enoyl-CoA complex is not observed. In addition, octanyol-CoA strongly protects the native enzyme against alkylation with iodoacetate. These results suggest that the methionine residue is within the active center of acyl-CoA dehydrogenase. Carboxymethylation of this residue may disrupt the precise orientation of the substrate required to achieve transfer of reducing equivalents to the flavin. Pig kidney general acyl-CoA dehydrogenase does not contain exposed catalytically essential cysteine residues.