Electron transfer, oxygen binding, and nitric oxide feedback inhibition inendothelial 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 s ynthase (eNOS) is regulated. Stopped-flow analysis of NADPH-dependent flavi n 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 di oxy species (Soret peak at 427 nm) whose formation and decay kinetics indic ate it can participate in NO synthesis. The kinetics of heme-NO complex for mation 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 substr ate. Heme-NO complex formation caused eNOS NADPH oxidation and citrulline s ynthesis 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 b y eNOS is primarily because of slow electron transfer from its reductase do main to the heme, rather than heme-NO complex formation or other aspects of catalysis. 2) eNOS forms relatively little heme-NO complex during NO synth esis from Arg, implying NO feedback inhibition has a minimal role. These pr operties distinguish eNOS from the other NOS isoforms and provide a foundat ion to better understand its role in physiology and pathology.