Cytoglobin
nitric oxide dioxygenase activity, heme protein as donor
Nitric-oxide dioxygenase function of human cytoglobin with cellular reductants and in rat hepatocytes.
Cytoglobin (Cygb) was investigated for its capacity to function as a NO dioxygenase (NOD) in vitro and in hepatocytes. Ascorbate and cytochrome b(5) were found to support a high NOD activity. Cygb-NOD activity shows respective K(m) values for ascorbate, cytochrome b(5), NO, and O(2) of 0.25 mm, 0.3 microm, 40 nm, and approximately 20 microm and achieves a k(cat) of 0.5 s(-1). Ascorbate and cytochrome b(5) reduce the oxidized Cygb-NOD intermediate with apparent second order rate constants of 1000 m(-1) s(-1) and 3 x 10(6) m(-1) s(-1), respectively. In rat hepatocytes engineered to express human Cygb, Cygb-NOD activity shows a similar k(cat) of 1.2 s(-1), a K(m)(NO) of 40 nm, and a k(cat)/K(m)(NO) (k'(NOD)) value of 3 x 10(7) m(-1) s(-1), demonstrating the efficiency of catalysis. NO inhibits the activity at [NO]/[O(2)] 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 b(5) as reductants.