Superoxide generated by glutathione reductase initiates a vanadate-dependent free radical chain oxidation of NADH

Abstract

Vanadate V (V) markedly stimulated the oxidation of NADPH by GSSG reductase and this oxidation was accompanied by the consumption of O 2 and the accumulation of H 2O 2. Superoxide dismutases completely eliminated this effect of V (V), whereas catalase was without effect, as was exogenous H 2O 2 added to 0.1 m m. These effects could be seen equally well in phosphate- or in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered solutions. Under anaerobic conditions there was no V (V)-stimulated oxidation of NADPH. Approximately 4% of the electrons flowing from NADPH to O 2, through GSSG reductase, resulted in release of O 2 −. The average length of the free radical chains causing the oxidation of NADPH, initiated by O 2 − plus V (V), was calculated to be in the range 140–200 NADPH oxidized per O 2 − introduced. We conclude that GSSG reductase, and by extension other O 2 −-producing flavoprotein dehydrogenases such as lipoyl dehydrogenase and ferredoxin reductase, catalyze V (V)-stimulated oxidation of NAD(P)H because they release O 2 − and because O 2 − plus V (V) initiate a free radical chain oxidation of NAD(P)H. There is no reason to suppose that these enzymes can act as NAD(P)H:V (V) oxidoreductases.