On the transhydrogenase activity of baker's yeast flavocytochrome b2

It is shown that, when baker's yeast flavocytochrome b2 is incubated with bromopyruvate in the presence of excess lactate, a transhydrogenation reaction takes place which produces bromolactate and pyruvate. The heme remains reduced during the reaction. It is further shown that reduced flavocytochrome b2 can catalyze the reduction of a number of other keto acids like pyruvate (the product of the physiological reaction) and other halogenopyruvates. Determinations of forward and reverse reaction rates, as well as of the redox potentials of the halogenolactate/halogenopyruvate couples lead to the conclusion that the transhydrogenation reaction is under thermodynamic control. Determinations of the steady-state deuterium isotope effect show that the rate-limiting step in the oxidation of halogenolactates is abstraction of the alpha-hydrogen (probably as a proton), as is the case for lactate itself. According to the principle of microscopic reversibility, the rate-limiting step in the reverse reaction must be protonation of the putative carbanion.