SUPEROXIDE PRODUCTION DURING REDUCTION OF MOLECULAR-OXYGEN BY ASSIMILATORY NITRATE REDUCTASE

Assimilatory NADH:nitrate reductase catalyzes the transfer of reducing equivalents from NADH to molecular oxygen. Initial rate studies perfo rmed under conditions of optimum pH (8.0) and constant ionic strength (mu = 0.2) revealed that the maximal rate of activity with molecular o xygen was 0.5% (0.044 mu mol NADH consumed/min/nmol heme) that of the activity using NO3- as the terminal electron acceptor (9.0 mu mol NADH consumed/min/nmol heme) with a K-m for O-2 of 586 mu M. NADH:molecula r oxygen reductase activity exhibited a pH optimum of 9.2, was inhibit ed by cyanide, and was unaffected by changes in ionic strength or the presence of phosphate ions, Spectroscopic studies indicated NADH:molec ular oxygen reductase activity resulted in the production of the super oxide radical, detected as the formation of adrenochrome from epinephr ine and by the reduction of nitroblue tetrazolium, both of which could be inhibited by the addition of superoxide dismutase and were unaffec ted by the addition of catalase, Direct observation of superoxide prod uction using spin-trapping in combination with EPR spectroscopy result ed in the detection of the spin adduct 5,5-dimethyl-5-hydroxy-1-pyrrol idinyloxy (DMPO-OH), The formation of this spin adduct was abolished e ither in the absence of nitrate reductase, NADH, or DMPO or by the add ition of superoxide dismutase or nitrate and was greatly reduced by th e presence of cyanide. Inclusion of catalase or ethanol had no effect on the formation of the spin adduct, These results indicate that nitra te reductase can utilize molecular oxygen as an electron acceptor and that the product, O-2(.-), is primarily generated via the Mopterin cen ter. (C) 1996 Academic Press, Inc.