Kinetic Studies on the Oxidation and Reduction of the Protoheme Moiety of Yeast L (+)-Lactate Dehydrogenase

Abstract

1. When the reduced form of yeast L (+)-lactate dehydrogenase [EC 1. 1. 2. 3] was oxidized by molecular oxygen or ferricyanide, an absorption spectrum having maxima at 533 and 567.5 mμ appeared. It was confirmed that the enzyme having the absorption maxima at 533 and 567.5 mμ is in the true oxidized form containing trivalent iron atoms. The difference absorption spectrum at the steady state of reaction with ferricyanide as acceptor, which was followed by the “stopped flow” method, was in agreement with that of reduced minus oxidized form of the enzyme.
2. The rate of reduction of the protoheme moiety was measured by the “stopped flow” method in the presence of substrate and acceptor.
Except in the case of methylene blue used as acceptor, the k^max_red/2-values (k^max_red: apparent rate constant of the reduction of protoheme at saturating concentrations of the substrate) at pH 7.2 showed a close agreement with those of the maximum rate (V_m/e) obtained by the overall reaction kinetics. The value of k^max_red/2 was markedly larger than that of V_m/e when methylene blue was used as acceptor. It was inferred that, except in the case of methylene blue and under the experimental conditions used, the acceptors receive electrons mainly through the protoheme moiety, whereas methylene blue accepts electrons partially from protoheme and partially from the reduced flavin moiety.
At pH 5.2, k^max_red/2=V_m/e when ferricyanide was used as acceptor, while k^max_red/2<V_m/e in the cases of other acceptors. This fact was construed as indicating that ferricyanide molecules can accept electrons only from the reduced protoheme, while at pH 5.2 other acceptors (methylene blue, thionine et_??_.) accept electrons mainly from the reduced flavin moiety.
3. From the relationship beetween k^max_red and pH, the pK value at 25°C was found to be 6.1 independently of the kind of acceptors used. -values, which were obtained
4. The k^max_red-values, which were obtained at pH 5.2 and 7.2 using ferricyanide as acceptor, were both found to be independent of the concentration of the enzyme added. The data obtained at pH 5.2 seemed to be in favor of the assumption that the electron-transfer from flavin to protoheme be an intramolecular reaction process.
5. The reaction scheme proposed in the preceding paper (1) was shown to be also capable of explaining various experimental facts observed in the present study.
The authors wish to express their gratitude to Prof. H. Tamiya for his valuable criticism in this work. Thanks are also due to Oriental Yeast Co. for the supply of baker's yeast.