Inactivation of two-electron reduced medium chain acyl-CoA dehydrogenase by 2-octynoyl-CoA.

The acetylenic thioester, 2-octynoyl-CoA, inactivates medium chain acyl-CoA dehydrogenase from pig kidney by two distinct pathways depending on the redox state of the FAD prosthetic group. Inactivation of the oxidized dehydrogenase occurs with labeling of an active site glutamate residue and elimination of CoASH. Incubation of the reduced dehydrogenase with 2-octynoyl-CoA rapidly forms a kinetically stable dihydroflavin species which is resistant to reoxidation using trans-2-octenoyl-CoA, molecular oxygen, or electron transferring flavoprotein. The reduced enzyme derivative shows extensive bleaching at 446 nm with shoulders at 320 and 380 nm. Denaturation of the reduced derivative in 80% methanol yields a mixture of products which was characterized by HPLC, by uv/vis, and by radiolabeling experiments. Approximately 20% of the flavin is recovered as oxidized FAD, about 40% is retained covalently attached to the protein, and the remainder is distributed between several species eluting after FAD on reverse-phase HPLC. The spectrum of one of these species ressembles that of a N(5)-C(4a) dihydroflavin adduct. These data suggest that a primary reduced flavin species undergoes various rearrangements during release from the protein. The possibility that the inactive modified enzyme represents a covalent adduct between 2-octynoyl-CoA and reduced flavin is discussed. Analogous experiments using enzyme substituted with 1,5-dihydro-5-deaza-FAD show rapid and quantitative reoxidation of the flavin by 0.5 eq of 2-octynoyl-CoA.