Electron transfer, oxygen binding, and nitric oxide feedback inhibition in endothelial nitric-oxide synthase.
We studied steps that make up the initial and steady-state phases of nitric oxide (NO) synthesis to understand how activity of bovine endothelial NO synthase (eNOS) is regulated. Stopped-flow analysis of NADPH-dependent flavin reduction showed the rate increased from 0. 13 to 86 s(-1) upon calmodulin binding, but this supported slow heme reduction in the presence of either Arg or N(omega)-hydroxy-l-arginine (0.005 and 0.014 s(-1), respectively, at 10 degrees C). O(2) binding to ferrous eNOS generated a transient ferrous dioxy species (Soret peak at 427 nm) whose formation and decay kinetics indicate it can participate in NO synthesis. The kinetics of heme-NO complex formation were characterized under anaerobic conditions and during the initial phase of NO synthesis. During catalysis heme-NO complex formation required buildup of relatively high solution NO concentrations (>50 nm), which were easily achieved with N(omega)-hydroxy-l-arginine but not with Arg as substrate. Heme-NO complex formation caused eNOS NADPH oxidation and citrulline synthesis to decrease 3-fold and the apparent K(m) for O(2) to increase 6-fold. Our main conclusions are: 1) The slow steady-state rate of NO synthesis by eNOS is primarily because of slow electron transfer from its reductase domain to the heme, rather than heme-NO complex formation or other aspects of catalysis. 2) eNOS forms relatively little heme-NO complex during NO synthesis from Arg, implying NO feedback inhibition has a minimal role. These properties distinguish eNOS from the other NOS isoforms and provide a foundation to better understand its role in physiology and pathology.
Abu-Soud
H M
HM
Department of Immunology, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA. abusouh@ccf.org
Ichimori
K
K
Presta
A
A
Stuehr
D J
DJ
D000818:Animals; D002147:Calmodulin / Q000494:pharmacology; D002417:Cattle; D003001:Cloning, Molecular; D004579:Electron Transport; D004926:Escherichia coli; D005246:Feedback; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III; D010100:Oxygen / Q000378:metabolism*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity
D002147:Calmodulin; D011994:Recombinant Proteins; D009569:Nitric Oxide; D006418:Heme; D009249:NADP; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D010100:Oxygen