NAD(P)H:quinone oxidoreductase (NQOI; EC 1.6.99.2) catalyzes a two-electron transfer involved in the protection of cells from reactive oxygen species. These reactive oxygen species are often generated by the one-electron reduction of quinones or quinone analogs. We report here on the previously unreported Fe(III) reduction activity of human NQOI. Under steady state conditions with Fe(III) citrate, the apparent Michaelis-Menten constant (K-m(app)) was similar to 0.3 nM and the apparent maximum velocity (V-max(app)) was 16 U mg(-1). Substrate inhibition was observed above 5 nM. NADH was the electron donor, K-m(app) 340 mu M and V-max(app) = 46 U mg(-1). FAD was also a cofactor with a K-m(app) of 3.1 mu M and V-max(app) of 89 U mg(-1). The turnover number for NADH oxidation was 25 s(-1). Possible physiological roles of the Fe(III) reduction by this enzyme are discussed. (c) 2006 Elsevier Inc. All rights reserved.