NAD(P)H:quinone oxidoreductase 1:: Role as a superoxide scavenger

Experiments using purified recombinant human NAD(P)H:quinone oxidoreductase 1 (NQO1) revealed that the auto-oxidation of fully reduced protein resulted in a 1: 1 stoichiometry of oxygen consumption to NADH oxidation with the production of hydrogen peroxide. The rate of auto-oxidation of fully reduced NQO1 was markedly accelerated in the presence of superoxide (O-2(.-)), whereas the addition of superoxide dismutase greatly inhibited the rate of auto-oxidation. The ability of reduced NQO1 to react with O-2(.-) suggested a role for NQO1 in scavenging O-2(.-), and this hypothesis was tested using established methods for O-2(.-) production and detection. The addition of NQO1 in combination with NAD( P) H resulted in inhibition of dihydroethidium oxidation, pyrogallol auto-oxidation, and elimination of a potassium superoxide-generated ethoxycarbonyl-2-methyl3,4-dihydro-2H-pyrrole-1-oxide:O-2(.-) adduct signal ( electron spin resonance). Kinetic parameters for the reduction of O-2(.-) by NQO1 were estimated using xanthine/xanthine oxidase as the source of O-2(.-) and after NQO1-dependent NADH oxidation at 340 nm. The ability of NQO1 to scavenge O-2(.-) was also examined using cell sonicates prepared from isogenic cell lines containing no NQO1 activity (NQO1(-)) or very high levels of NQO1 activity (NQO1(+)). We demonstrated that addition of NAD(P)H and cell sonicate from NQO1(+) but not NQO1(-) cells resulted in an increased level of O-2(.-) scavenging, and this increased level of O-2(.-) scavenging could be inhibited by 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione (ES936), a mechanism-based inhibitor of NQO1. NQO1 can generate hydroquinones that are redox active, and the O-2(.-) scavenging activity of NQO1 may allow protection against O-2(.-) at the site of hydroquinone generation. In addition, the O-2(.-) scavenging activity of NQO1 may provide an additional level of protection against O-2(.-)-induced toxicity.