PT - JOURNAL ARTICLE AU - Birte Plitzko AU - Antje Havemeyer AU - Bettina Bork AU - Florian Bittner AU - Ralf Mendel AU - Bernd Clement TI - Defining the Role of the NADH–Cytochrome-<em>b</em><sub>5</sub> Reductase 3 in the Mitochondrial Amidoxime Reducing Component Enzyme System AID - 10.1124/dmd.116.071845 DP - 2016 Oct 01 TA - Drug Metabolism and Disposition PG - 1617--1621 VI - 44 IP - 10 4099 - http://dmd.aspetjournals.org/content/44/10/1617.short 4100 - http://dmd.aspetjournals.org/content/44/10/1617.full SO - Drug Metab Dispos2016 Oct 01; 44 AB - The importance of the mitochondrial amidoxime reducing component (mARC)–containing enzyme system in N-reductive metabolism has been studied extensively. It catalyzes the reduction of various N-hydroxylated compounds and therefore acts as the counterpart of cytochrome P450– and flavin-containing monooxygenase–catalyzed oxidations at nitrogen centers. This enzyme system was found to be responsible for the activation of amidoxime and N-hydroxyguanidine prodrugs in drug metabolism. The synergy of three components (mARC, cytochrome b5, and the appropriate reductase) is crucial to exert the N-reductive catalytic effect. Previous studies have demonstrated the involvement of the specific isoforms of the molybdoenzyme mARC and the electron transport protein cytochrome b5 in N-reductive metabolism. To date, the corresponding reductase involved in N-reductive metabolism has yet to be defined because previous investigations have presented ambiguous results. Using small interfering RNA–mediated knockdown in human cells and assessing the stoichiometry of the enzyme system reconstituted in vitro, we provide evidence that NADH–cytochrome-b5 reductase 3 is the principal reductase involved in the mARC enzyme system and is an essential component of N-reductive metabolism in human cells. In addition, only minimal levels of cytochrome-b5 reductase 3 protein are sufficient for catalysis, which impeded previous attempts to identify the reductase.