Geminate recombination of nitric oxide to endothelial nitric-oxide synthase and mechanistic implications.
The nitric-oxide synthase (NOS) catalyzes the oxidation of L-arginine to L-citrulline and NO through consumption of oxygen bound to the heme. Because NO is produced close to the heme and may bind to it, its subsequent role in a regulatory mechanism should be scrutinized. We therefore examined the kinetics of NO rebinding after photodissociation in the heme pocket of human endothelial NOS by means of time-resolved absorption spectroscopy. We show that geminate recombination of NO indeed occurs and that this process is strongly modulated by L-Arg. This NO rebinding occurs in a multiphasic fashion and spans over 3 orders of magnitude. In both ferric and ferrous states of the heme, a fast nonexponential picosecond geminate rebinding first takes place followed by a slower nanosecond phase. The rates of both phases decreased, whereas their relative amplitudes are changed by the presence of L-Arg; the overall effect is a slow down of NO rebinding. For the isolated oxygenase domain, the picosecond rate is unchanged, but the relative amplitude of the nanosecond binding decreased. We assigned the nanosecond kinetic component to the rebinding of NO that is still located in the protein core but not in the heme pocket. The implications for a mechanism of regulation involving NO binding are discussed.
Négrerie
M
M
INSERM U451, Ecole Nationale Supérieure des Techniques Avancées Centre de l'Yvette, Chemin de la Hunière, 91761 Palaiseau Cedex, France. negrerie@enstay.ensta.fr
Berka
V
V
Vos
M H
MH
Liebl
U
U
Lambry
J C
JC
Tsai
A L
AL
Martin
J L
JL
D001120:Arginine / Q000737:chemistry; D001708:Biopterins / Q000031:analogs & derivatives / Q000737:chemistry; D006418:Heme / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D009569:Nitric Oxide / Q000737:chemistry*; D019001:Nitric Oxide Synthase / Q000737:chemistry*; D052250:Nitric Oxide Synthase Type III; D010782:Photolysis; D011994:Recombinant Proteins / Q000737:chemistry; D013057:Spectrum Analysis / Q000379:methods
D011994:Recombinant Proteins; D001708:Biopterins; D009569:Nitric Oxide; D006418:Heme; D001120:Arginine; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C003402:sapropterin