Detection of NAD(P)H--rubredoxin oxidoreductases in Clostridia

Rubredoxin mediates the electron flow from NAD(P)H-rubredoxin oxidoreductase to metmyoglobin. Metmyoglobin is a good electron acceptor of the reduced rubredoxin and a poor electron acceptor of the NAD(P)H diaphorase activities from the clostridial extracts; these properties allow detecting and measuring NAD(P)H-rubredoxin oxidoreductase activities in crude extracts of Clostridia. The metmyoglobin reduction is quantitatively determined by spectrophotometric measurements at 581 nm. The rate of metmyoglobin reduction is constant for at least 3 min in the presence of 0.1 to 0.6 mg/ml of extract from Clostridium acetobutylicum and 0.05–0.5 nmol/ml of rubredoxin. In C. pasteurianum and C. tyrobutyricum measurement of the rubredoxin chromophore reduction by crude extracts in the presence of NAD(P)H requires a large amount of rubredoxin; these unphysiological concentrations allow unspecific enzymatic reactions which lead to erroneous interpretations.