The oxidation of reduced putidaredoxin reductase by oxidized putidaredoxin.

NAD+ was shown to be essential for the kinetically favored oxidation of putidaredoxin reductase from the two electron reduced form to the semiquinone form by oxidized putidaredoxin. The rate of oxidation of putidaredoxin reductase from the semiquinone to the oxidized form was not commensurate with the overall rate of catalysis seen in the cytochrome c reductase assay. The presence of excess NADH was shown to result in a substantial enhancement of the rate of the catalytic cycle. Thus, pyridine nucleotides play an important role in the reoxidation of putidaredoxin reductase by putidaredoxin. The initial velocity patterns for the addition of varying concentrations of substrates indicate a ping-pong mechanism for substrate addition while product inhibition patterns indicate otherwise. A model of putidaredoxin reductase catalysis is described which accounts for the product inhibition patterns and the observed role of pyridine nucleotides in the reaction cycle. The two site mechanism described in this manuscript has both sequential and ping-pong features which are accounted for by the two addition steps of oxidized putidaredoxin during catalytic turnover.