Nitric-oxide Dioxygenase Function of Human Cytoglobin with Cellular Reductants and in Rat Hepatocytes

Abstract Cytoglobin (Cygb) was investigated for its capacity to function as a NO dioxygenase (NOD) in vitro and in hepatocytes. Ascorbate and cytochrome b5 were found to support a high NOD activity. Cygb-NOD activity shows respective Km values for ascorbate, cytochrome b5, NO, and O2 of 0.25 mm, 0.3 μm, 40 nm, and ∼20 μm and achieves a kcat of 0.5 s−1. Ascorbate and cytochrome b5 reduce the oxidized Cygb-NOD intermediate with apparent second order rate constants of 1000 m−1 s−1 and 3 × 106 m−1 s−1, respectively. In rat hepatocytes engineered to express human Cygb, Cygb-NOD activity shows a similar kcat of 1.2 s−1, a Km(NO) of 40 nm, and a kcat/Km(NO) (k′NOD) value of 3 × 107 m−1 s−1, demonstrating the efficiency of catalysis. NO inhibits the activity at [NO]/[O2] ratios >1:500 and limits catalytic turnover. The activity is competitively inhibited by CO, is slowly inactivated by cyanide, and is distinct from the microsomal NOD activity. Cygb-NOD provides protection to the NO-sensitive aconitase. The results define the NOD function of Cygb and demonstrate roles for ascorbate and cytochrome b5 as reductants.