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.
Gardner
Anne M
AM
Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229, USA.
Cook
Matthew R
MR
Gardner
Paul R
PR
D000818:Animals; D001205:Ascorbic Acid / Q000737:chemistry; D018938:Caco-2 Cells; D002384:Catalysis; D045744:Cell Line, Tumor; D015786:Cytochromes b5 / Q000737:chemistry; D000078842:Cytoglobin; D005914:Globins / Q000737:chemistry / Q000378:metabolism*; D006418:Heme / Q000737:chemistry; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D009419:Nerve Tissue Proteins / Q000737:chemistry; D000078787:Neuroglobin; D009569:Nitric Oxide / Q000737:chemistry; D010105:Oxygenases / Q000378:metabolism*; D051381:Rats
C000627265:CYGB protein, human; C000627268:Cygb protein, rat; D000078842:Cytoglobin; D009419:Nerve Tissue Proteins; D000078787:Neuroglobin; D009569:Nitric Oxide; D006418:Heme; D005914:Globins; D015786:Cytochromes b5; D010105:Oxygenases; C114415:nitric oxide dioxygenase; D001205:Ascorbic Acid