Mitochondrial amidoxime reducing component (mARC) proteins are molybdopterin-containing enzymes of unclear physiological function. Both human isoforms mARC-1 and mARC-2 are able to catalyze the reduction of nitrite when they are in the reduced form. Moreover, our results indicate that mARC can generate nitric oxide (NO) from nitrite when forming an electron transfer chain with NADH, cytochrome b₅, and NADH-dependent cytochrome b₅ reductase. The rate of NO formation increases almost 3-fold when pH was lowered from 7.5 to 6.5. To determine if nitrite reduction is catalyzed by molybdenum in the active site of mARC-1, we mutated the putative active site cysteine residue (Cys-273), known to coordinate molybdenum binding. NO formation was abolished by the C273A mutation in mARC-1. Supplementation of transformed Escherichia coli with tungsten facilitated the replacement of molybdenum in recombinant mARC-1 and abolished NO formation. Therefore, we conclude that human mARC-1 and mARC-2 are capable of catalyzing reduction of nitrite to NO through reaction with its molybdenum cofactor. Finally, expression of mARC-1 in HEK cells using a lentivirus vector was used to confirm cellular nitrite reduction to NO. A comparison of NO formation profiles between mARC and xanthine oxidase reveals similar K_cat and V_max values but more sustained NO formation from mARC, possibly because it is not vulnerable to autoinhibition via molybdenum desulfuration. The reduction of nitrite by mARC in the mitochondria may represent a new signaling pathway for NADH-dependent hypoxic NO production.

Aims: Recent studies suggest that the molybdenum enzymes xanthine oxidase, aldehyde oxidase, and mARC exhibit nitrite reductase activity at low oxygen pressures. However, inhibition studies of xanthine oxidase in humans have failed to block nitrite-dependent changes in blood flow, leading to continued exploration for other candidate nitrite reductases. Another physiologically important molybdenum enzyme—sulfite oxidase (SO)—has not been extensively studied. Results: Using gas-phase nitric oxide (NO) detection and physiological concentrations of nitrite, SO functions as nitrite reductase in the presence of a one-electron donor, exhibiting redox coupling of substrate oxidation and nitrite reduction to form NO. With sulfite, the physiological substrate, SO only facilitates one turnover of nitrite reduction. Studies with recombinant heme and molybdenum domains of SO indicate that nitrite reduction occurs at the molybdenum center via coupled oxidation of Mo(IV) to Mo(V). Reaction rates of nitrite to NO decreased in the presence of a functional heme domain, mediated by steric and redox effects of this domain. Using knockdown of all molybdopterin enzymes and SO in fibroblasts isolated from patients with genetic deficiencies of molybdenum cofactor and SO, respectively, SO was found to significantly contribute to hypoxic nitrite signaling as demonstrated by activation of the canonical NO-sGC-cGMP pathway. Innovation: Nitrite binds to and is reduced at the molybdenum site of mammalian SO, which may be allosterically regulated by heme and molybdenum domain interactions, and contributes to the mammalian nitrate-nitrite-NO signaling pathway in human fibroblasts. Conclusion: SO is a putative mammalian nitrite reductase, catalyzing nitrite reduction at the Mo(IV) center. Antioxid. Redox Signal. 23, 283–294.