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 neuronal nitric oxide synthase (nNOS) reductase domain, which contains the FAD-FMN prosthetic group pair and calmodulin-binding site, catalyzed aerobic NADPH-oxidation in the presence of the model quinone compound menadione (MD), including antitumor mitomycin C (Mit C) and adriamycin (Adr). Calcium/calmodulin (Ca2+/CaM) stimulated the NADPH oxidation of these quinones. The MD-mediated NADPH oxidation was inhibited in the presence of NAD(P)H:quinone oxidoreductase (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 quinones. In the presence or absence of Ca2+/CaM, the semiquinone radical species were major intermediates observed during the oxidation of the reduced enzyme by MD, but the fully reduced flavin species did not significantly accumulate under these conditions. Air-stable semiquinone did not react rapidly with MD, but the fully reduced species of both flavins, FAD and FMN, could donate one electron to MD. The intramolecular electron transfer between the two flavins is the rate-limiting step in the catalytic cycle [H. Matsuda, T. Iyanagi, Biochim. Biophys. Acta 1473 (1999) 345-355). These data suggest that the enzyme functions between the 1e- <==> 3e- level during one-electron reduction of MD, and that the rates of quinone reductions are stimulated by a rapid electron exchange between the two flavins in the presence of Ca2+/CaM.
Matsuda
H
H
Department of Life Science, Faculty of Science, Himeji, Institute of Technology, Harima Science Garden City, Hyogo, Japan.
Kimura
S
S
Iyanagi
T
T
D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry; D004317:Doxorubicin / Q000737:chemistry; D004583:Electrons; D005292:Ferricyanides / Q000737:chemistry; D005415:Flavins / Q000737:chemistry; D007700:Kinetics; D016685:Mitomycin / Q000737:chemistry; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D019001:Nitric Oxide Synthase / Q000737:chemistry*; D052248:Nitric Oxide Synthase Type I; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry; D011809:Quinones / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D014812:Vitamin K / Q000737:chemistry
D005292:Ferricyanides; D005415:Flavins; D009419:Nerve Tissue Proteins; D011809:Quinones; D011994:Recombinant Proteins; D014812:Vitamin K; C007931:hexacyanoferrate III; D016685:Mitomycin; D004317:Doxorubicin; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; C080610:nitric-oxide reductase (P450)