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-methyl-3,4-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 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.
Siegel
David
D
Department of Pharmaceutical Sciences and Cancer Center, School of Pharmacy, University of Colorado Health Sciences Center, 4200 East 9th Ave., Denver, CO 80262, USA.
Gustafson
Daniel L
DL
Dehn
Donna L
DL
Han
Jin Yi
JY
Boonchoong
Preecha
P
Berliner
Lawrence J
LJ
Ross
David
D
D000818:Animals; D016466:CHO Cells; D002455:Cell Division / Q000187:drug effects; D006224:Cricetinae; D004578:Electron Spin Resonance Spectroscopy; D004996:Ethidium / Q000031:analogs & derivatives* / Q000378:metabolism; D016166:Free Radical Scavengers / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D010084:Oxidation-Reduction; D011748:Pyrogallol / Q000378:metabolism / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*; D014162:Transfection
D016166:Free Radical Scavengers; D017382:Reactive Oxygen Species; D011748:Pyrogallol; C067883:dihydroethidium; D013481:Superoxides; D016660:NAD(P)H Dehydrogenase (Quinone); C447479:NQO1 protein, human; D004996:Ethidium