Nitrite reductase and nitric-oxide synthase activity of the mitochondrial molybdopterin enzymes mARC1 and mARC2.
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 Kcat and Vmax 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.
Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15261; Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213.
Courtney E
CE
Sparacino-Watkins
Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15261.
Jesús
J
Tejero
Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15261; Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213.
Bin
B
Sun
Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15261; Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213.
Marc C
MC
Gauthier
Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, Pennsylvania 15282.
John
J
Thomas
Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15261.
Venkata
V
Ragireddy
Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15261.
Bonnie A
BA
Merchant
Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15261.
Jun
J
Wang
Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15261.
Ivan
I
Azarov
Department of Chemistry and Biochemistry, Duquesne University, Pittsburgh, Pennsylvania 15282.
Partha
P
Basu
Vascular Medicine Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15261; Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213. Electronic address: gladwinmt@upmc.edu.
Mark T
MT
Gladwin
D000595:Amino Acid Sequence; D003067:Coenzymes / Q000378:metabolism*; D003579:Cytochrome Reductases / Q000378:metabolism; D015786:Cytochromes b5 / Q000378:metabolism; D004579:Electron Transport; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008667:Metalloproteins / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology*; D024101:Mitochondrial Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D008982:Molybdenum / Q000378:metabolism; D000090006:Molybdenum Cofactors; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D009572:Nitrite Reductases / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D011621:Pteridines / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014969:Xanthine Oxidase / Q000378:metabolism
D003067:Coenzymes; D003579:Cytochrome Reductases; D015786:Cytochromes b5; D008667:Metalloproteins; D024101:Mitochondrial Proteins; D008982:Molybdenum; D000090006:Molybdenum Cofactors; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D009572:Nitrite Reductases; D009573:Nitrites; D010088:Oxidoreductases; D010100:Oxygen; D011621:Pteridines; D011994:Recombinant Proteins; D014969:Xanthine Oxidase; C022261:molybdopterin; C554553:MTARC1 protein, human; C554552:mitochondrial amidoxime reducing component 2, human
Hypoxia; Mitochondria; Molybdenum; Nitric Oxide; Reductase; Vascular Biology