flavin-adenine-dinucleotide and ferrocenium

The intense charge transfer complex between the enolate of 3-thia-octanoyl-CoA and the oxidized flavin of the medium-chain acyl-CoA dehydrogenase is discharged by the ferricenium ion with irreversible inactivation of the enzyme. Charge transfer complex formation is a necessary, but insufficient, condition for oxidative inactivation: the 3-oxa-octanoyl-CoA complex is also inactivated, whereas the comparable trans-3-octenoyl-CoA species is not. Complete inactivation of the dehydrogenase with 3-thia-octanoyl-CoA requires 1 molecule of thioester and apparently 3 molecules of ferricenium hexafluorophosphate. Experiments with 8-Cl-FAD substituted enzyme and the crystal structure of enzyme.ligand complexes argue that ferricenium ion-mediated oxidation proceeds through the flavin prosthetic group. Synthesis of [2-14C]-3-thia-octanoyl-CoA, followed by isolation of radiolabeled peptide from the modified medium-chain dehydrogenase, showed that inactivation results in labeling the catalytic base, GLU376. Oxidative modification is accompanied by the release of CoASH. A mechanism for inactivation is proposed involving generation of a sulfonium salt which efficiently captures the carboxylate nucleophile.

Topics: Acyl Coenzyme A; Acyl-CoA Dehydrogenase; Acyl-CoA Dehydrogenases; Affinity Labels; Amino Acid Sequence; Binding Sites; Coenzyme A; Enzyme Inhibitors; Esters; Ferrous Compounds; Flavin-Adenine Dinucleotide; Flavoproteins; Ligands; Models, Chemical; Molecular Sequence Data; Oxidation-Reduction; Peptide Fragments; Sequence Analysis; Spectrophotometry; Sulfhydryl Compounds