1. The rates of oxidation and reduction of FMN and protoheme, which are the prosthetic groups of L(+)-lactate dehydrogenase [EC 1. 1. 2. 3, L-lactate: ferricytochrome c oxidoreductase], were estimated spectrophotometrically by the “stopped flow method.” The rates of reduction of these prosthetic groups were measured also with the “continuous flow method” by mixing the oxidized enzyme with a solution of the substrate under anaerobic conditions. The values obtained were compared with that of the rate of the overall reaction.
2. Although no formation of a semiquinone of the bound FMN was observed in the steady state at a sufficient concentration of ferricyanide, it was found that the semiquinone of the bound FMN can be produced very rapidly when the enzyme solution (oxidized form) is mixed with the substrate in the absence of electron acceptor under anaerobic conditions. It was suggested that the enzyme having the semiquinone form of the bound FMN is an active intermediate which appears during the reaction.
3. Assuming that an electron acceptor can react directly with the reduced protoheme but not with the reduced FMN, a mechanism of this enzyme reaction was proposed. On the basis of the kinetic data, it was concluded that the rate-limiting step in the overall reaction is the hydrogen transfer from the substrate attached to the enzyme to the bound FMN, and that the intramolecular electron transfer from the reduced FMN (FMNH₂ or FMNH.) to the oxidized protoheme is very rapid.