Naphthoquinone-dependent generation of superoxide radicals by quinone reductase isolated from the plasma membrane of soybean.
Using a tetrazolium-based assay, a NAD(P)H oxidoreductase was purified from plasma membranes prepared from soybean (Glycine max) hypocotyls. The enzyme, a tetramer of 85 kD, produces O2(.-) by a reaction that depended on menadione or several other 1,4-naphthoquinones, in apparent agreement with a classification as a one-electron-transferring flavoenzyme producing semiquinone radicals. However, the enzyme displayed catalytic and molecular properties of obligatory two-electron-transferring quinone reductases of the DT-diaphorase type, including insensitivity to inhibition by diphenyleneiodonium. This apparent discrepancy was clarified by investigating the pH-dependent reactivity of menadionehydroquinone toward O2 and identifying the protein by mass spectrometry and immunological techniques. The enzyme turned out to be a classical NAD(P)H:quinone-acceptor oxidoreductase (EC 1.6.5.2, formerly 1.6.99.2) that reduces menadione to menadionehydroquinone and subsequently undergoes autoxidation at pH > or = 6.5. Autoxidation involves the production of the semiquinone as an intermediate, creating the conditions for one-electron reduction of O2. The possible function of this enzyme in the generation of O2(.-) and H2O2 at the plasma membrane of plants in vivo is discussed.
Schopfer
Peter
P
Universität Freiburg, Institut für Biologie II, D-79104 Freiburg, Germany.
Heyno
Eiri
E
Drepper
Friedel
F
Krieger-Liszkay
Anja
A
D000595:Amino Acid Sequence; D015153:Blotting, Western; D002384:Catalysis; D002462:Cell Membrane / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008969:Molecular Sequence Data; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000378:metabolism; D009285:Naphthoquinones / Q000378:metabolism*; D010084:Oxidation-Reduction; D013025:Glycine max / Q000201:enzymology*; D013481:Superoxides / Q000378:metabolism*
D004791:Enzyme Inhibitors; D009285:Naphthoquinones; D013481:Superoxides; D009247:NADH, NADPH Oxidoreductases; D016660:NAD(P)H Dehydrogenase (Quinone)