One-electron reduction of quinones by the neuronal nitric-oxide synthase reductase domain

Flavin electron transferases can catalyze one- or two-electron reduction of quinones including bioreductive antitumor quinones. The recombinant neuron al nitric oxide synthase (nNOS) reductase domain, which contains the FAD-FM N prosthetic group pair and calmodulin-binding site, catalyzed aerobic NADP H-oxidation in the presence of the model quinone compound menadione (MD), i ncluding antitumor mitomycin C (Mit C) and adriamycin (Adr), Calcium/calmod ulin (Ca2+/CaM) stimulated the NADPH oxidation of these quinones. The MD-me diated NADPH oxidation was inhibited in the presence of NAD(P)H:quinone oxi doreductase (QR), but Mit C- and Adr-mediated NADPH oxidations were not. In anaerobic conditions, cytochrome b5 as a scavenger for the menasemiquinone radical (MD.-) was stoichiometrically reduced by the nNOS reductase domain in the presence of MD, but not of QR. These results indicate that the nNOS reductase domain can catalyze a only one-electron reduction of bivalent qu inones. In the presence or absence of Ca2+/CaM, the semiquinone radical spe cies were major intermediates observed during the oxidation of the reduced enzyme by MD, but the fully reduced flavin species did not significantly ac cumulate under these conditions. Air-stable semiquinone did not react rapid ly with MD, but the fully reduced species of both flavins, FAD and FMN, cou ld donate one electron to MD, The intramolecular electron transfer between the two flavins is the rate-limiting step in the catalytic cycle [H. Matsud a, T. Iyanagi, Biochim. Biophys. Acta 1473 (1999) 345-355). These data sugg est that the enzyme functions between the 1e(-) reversible arrow 3e(-) leve l during one-electron reduction of MD, and that the rates of quinone reduct ions are stimulated by a rapid electron exchange between the two flavins in the presence of Ca2+/CaM. (C) 2000 Elsevier Science B.V. All rights reserv ed.