Flavoenzymes Reduce Vanadium(V) and Molecular Ozygen and Generate Hydroxyl Radical

"The mechanism of vanadium(V) V ) toxicity is still not fully understood in spite of extensive current effort (1-3). The presence of a vanadium(V) V ) -dependent NAD ( P )H oxidase in cell membranes suggests that at least one of the pathways involves oxidation of NAD(P)H (1-3). Considerable effort has therefore been devoted to finding the mechanism of NAD ( P ) H-oxidation by va¬nadium( V ) (1-19). Recently Liochev and Fridovich (4, 16) provided a critical review of vanadium( V) -stimulated NAD (P )H oxidation by plasma membranes. They out¬lined a mechanism in which vanadium(V) V )-stimulated oxidation of NAD ( P )H by biomembranes involves an O2 - initiated free radical chain reaction (4 ). While the above may be the major pathway for the vanadium-me¬diated NAD (P )H oxidation in membrane-related sys¬tems, some recent results suggest that in the presence of NADPH the flavoenzyme glutathione reductase can function as a vanadium(V) V ) reductase (18). Because of the significance of this result in relationship to the mech¬anism of vanadium( V) metabolism and the novelty of the metal-reductase behavior of a flavoenzyme, we have car¬ried out additional studies on one-electron reduction of vanadium( V) by two other, related flavoenzymes: lipoyl dehydrogenase and ferredoxin-NADPH oxidoreductase. These two enzymes were selected because they are known to catalyze a one-electron reduction of Cr ( VI ) (19-21 ), which is isoelectronic with vanadium( V ).The present study also reports on the generation of hydroxyl ( • OH) radicals in the vanadium( V) -flavoen-zyme redox system without exogenous H2O2. The • OH study was undertaken because • OH radicals are known to be generated in reactions of vanadium( IV) with H2O2 (17,18,22-27). For example, Piette and co-workers (17, 22) have studied effects of vanadium(IV) IV ) on lipid per-oxidation and NADPH oxidation and suggested that va-nadium( IV) -induced • OH radical formation may play an important role in vanadium( V) toxicity. Carmichael (25, 26) investigated the reaction between a RNA /VO 2' complex and H2O2 with a view to probe the possible re-lationship between vanadium( IV) -mediated • OH radical generation and DNA damage. He has suggested that in this reaction the • OH radical adds to the C (5) carbon of uracil, causing nucleotide damage, similar to what occurs in the nucleotide /Fe2+ / H2O2 reaction (28) and in y-irradiated aqueous solutions of nucleic acids (29 ). To our knowledge, however, there is as yet no report on ·OH radical generation in any reaction involving the reduction of vanadium ( V) by a well-characterized enzyme, without exogenous H20 2 • The present study provides evidence that ·OH radicals can form in a flavoenzyme-mediated reduction of vanadium( V) to vanadium( IV) and that this process involves also the reduction of molecular oxygen ( 0 2 ) to H20 2 • The H20 2 thus generated reacts with vanadium( IV) to produce ·OH radical, in analogy with the Fenton reaction ( Fe2 + + H20 2 - Fe3 + + OH- + ·OH)."