Kinetic Characterization of Nitrite Reduction to NO by the Molybdopterin Enzyme mARC2

Authors: Eric Cecco

Advisor: Dr. Courtney Sparacino-Watkins

Nitrite is a nitric oxide (NO) precursor that is endogenously produced in human tissues. Endothelial dysfunction involved in cardiovascular disease is characterized by a decreased NO production in the vascular endothelium. New therapeutic strategies using nitrite can enhance NO levels and potentially treat endothelial dysfunction. However, the mechanisms involved in NO production are incompletely defined and the Enzyme(s) responsible for biological activation of nitrite vary in each tissue.
Our lab identified a novel human nitrite-dependent NO synthase, the mitochondrial amidoxime reducing component (mARC) enzymes. Human mARC-1 and mARC-2 are molybdenum (Mo)-dependent oxidoreductases that function as part of a three-enzyme metabolon with cytochrome b5 (CYB5) and cytochrome b5 reductase (CYB5R). We hypothesize that the mARC-2 enzyme metabolon would exhibit similar kinetic characteristics to that of previously characterized mammalian molybdenum-dependent nitrite reductases. To test this hypothesis, we used isolated recombinant human mARC-2, CYB5, and CYB5R enzymes and NO-production rate measurements to determine (1) for affinity for nitrite, and (2) effect of pH.