On the mechanism of flavin modification during inactivation of flavocytochrome b2 from baker's yeast by acetylenic substrates.

The reaction of 2-hydroxy-3-butynoate, a suicide substrate, with flavocytochrome b2 [F. Lederer (1974) Eur. J. Biochem. 88, 393-399] has been reinvestigated. It is shown that no inactivation occurs under anaerobic conditions. In the presence of ferricyanide, the partition ratio between oxidation and inactivation is 3200. Ketobutynoate has no effect on oxidized flavocytochrome b2. But it inactivates the reduced enzyme, while undergoing catalytic reduction to hydroxybutynoate. The partition ratio between reduction and inactivation is 5. Inactivation followed by borohydride reduction was carried out in parallel with lactate oxidase and flavocytochrome b2. The decomposition products of the initial adduct formed between flavin and inactivator were isolated and characterized. One of them (compound II) was obtained from both enzymes and is identical to the one previously isolated from hydroxybutynoate-inactivated lactate oxidase [Schonbrunn et al. (1976) Biochemistry 15, 1798-1807]. Its decarboxylated derivative (compound I) was also formed. Another major adduct, compound III, was isolated only from flavocytochrome b2. Its structure and the conditions in which it appears suggest it is formed from the same primary adduct as compounds I and II, but by a different decomposition mode, on the enzyme itself. Altogether these results strengthen the idea that inactivation is caused by reaction between oxidized flavin and an allenic carbanion, the isomerisation product of a normal reaction intermediate. It is proposed that differences in the rate-determining step of the redox reaction explain the differences in the inactivation process which are observed between flavocytochrome b2, lactate oxidase and hydroxyacid oxidase.