Oxidation of NG-hydroxy-L-arginine by nitric oxide synthase: evidence for the involvement of the heme in catalysis.
The involvement of the protoporphyrin IX heme iron of macrophage nitric oxide synthase (NOS) in the oxidation of NG-hydroxy-L-arginine (L-NHA) to nitric oxide (NO) and citrulline was investigated by carbon monoxide (CO) inhibition studies and binding difference spectroscopy. A CO:oxygen mixture (80:20) was found to inhibit the reaction by 33% with L-NHA as a substrate compared to 57% with L-arginine. Spectral perturbations were observed upon the addition of L-NHA to oxidized NOS, producing a type I binding difference spectrum with a maximum at 384 nm and minimum at 420 nm. In addition, L-NHA was incapable of reducing anaerobic oxidized NOS in the absence of NADPH. These studies support the involvement of the heme in the oxidation of L-NHA to NO and citrulline, indicating that the heme functions in both of the currently characterized oxidative steps of the NOS reaction.
Pufahl
R A
RA
Department of Biological Chemistry, School of Medicine, University of Michigan, Ann Arbor 48109-1065.
Marletta
M A
MA
D000594:Amino Acid Oxidoreductases / Q000378:metabolism*; D000693:Anaerobiosis; D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives* / Q000378:metabolism; D002248:Carbon Monoxide / Q000494:pharmacology; D002384:Catalysis; D002460:Cell Line; D006418:Heme / Q000378:metabolism*; D007700:Kinetics; D008264:Macrophages / Q000201:enzymology; D051379:Mice; D019001:Nitric Oxide Synthase; D010084:Oxidation-Reduction; D013053:Spectrophotometry; D013379:Substrate Specificity
C007985:N(5)-hydroxy-L-arginine; D006418:Heme; D002248:Carbon Monoxide; D001120:Arginine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases