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 O-2(center dot-) 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-electrontransferring 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 O-2 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 >= 6.5. Autoxidation involves the production of the semiquinone as an intermediate, creating the conditions for one-electron reduction of O-2. The possible function of this enzyme in the generation of O-2(center dot-) and H2O2 at the plasma membrane of plants in vivo is discussed.