Nitric oxide complexes of inducible nitric oxide synthase: spectral characterization and effect on catalytic activity.
Nitric oxide synthase (NOS) catalyzes the oxidation of L-arginine to citrulline and nitric oxide (.NO). NOS is a hemoprotein containing a cytochrome P-450-type heme that has been shown to be involved in catalysis. It has been suggested that .NO is able to bind tightly to the heme of NOS and may in this way serve to regulate enzymatic activity. We report here the formation of both ferric and ferrous heme nitrosyl complexes with the inducible NOS from murine macrophages. The ferric nitrosyl complex is characterized by a Soret peak at 443 nm and two distinct peaks in the alpha/beta region at 549 and 585 nm. The ferrous nitrosyl complex has absorbance maxima at 436 and 566 nm. A transient spectral intermediate is observed under conditions of NOS turnover. This intermediate appears to be a mixture of ferric and ferrous nitrosyl complexes and is unstable in the presence of oxygen. Binding of L-arginine decreases the affinity of .NO for the ferric heme but does not appear to decrease the affinity of .NO for the ferrous heme. Addition of either oxyhemoglobin or methemoglobin to NOS assays results in a nearly 2-fold increase in enzymatic activity. This result is attributed to the ability of both forms of hemoglobin to decrease the concentration of .NO in solution and is consistent with .NO inhibition of NOS under assay conditions. Our results show that NOS nitrosyl complexes form under certain conditions but suggest that the relevance of such complexes to activity in vivo may be limited by their instability in an aerobic environment.
Hurshman
A R
AR
Interdepartmental Program in Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor 48109-1065, USA.
Marletta
M A
MA
D000594:Amino Acid Oxidoreductases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D000693:Anaerobiosis; D000818:Animals; D001120:Arginine / Q000378:metabolism; D002460:Cell Line; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000378:metabolism; D004790:Enzyme Induction; D006418:Heme / Q000378:metabolism; D006454:Hemoglobins / Q000494:pharmacology; D007700:Kinetics; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase; D013053:Spectrophotometry
D006454:Hemoglobins; D009569:Nitric Oxide; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D001120:Arginine; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases