Electron transferase activity of diaphorase (NADH: acceptor oxidoreductase) from Bacillus stearothermophilus

Electrochemical kinetic measurements were carried out for electron-transfer between NADH and the oxidized forms of mediators (ferrocenylmethanol (FMA), ferrocenyl-1-ethanol (FEA), N,N,N',N'-tetramethylphenylenediamine (TMPD), Co(Phen)2+(3) and Fe(CN)4-(6)) catalyzed by diaphorase (NADH: acceptor oxidoreductase, EC 1.6.99.-) purified from Bacillus stearothermophilus. Cyclic voltammograms for the mediators with excess NADH in the presence of diaphorase gave steady-state currents. The quantitative analysis of the dependence of the current on the mediator concentration yielded a Michaelis constant (Km) and molecular activity (ko), which are difficult to determine by the conventional spectrophotometric method. Small Km and large ko values were observed for the oxidized forms of FMA, FEA and TMPD compared to those for Co(Phen)3+(3) and Fe(CN)3-(6). It is suggested that the reaction pocket of the present diaphorase is hydrophobic. The present electrochemical procedure for the determination of the kinetic parameters is applicable widely to similar enzyme reactions.