Nitric Oxide (NO) Traffic in Endothelial NO Synthase

Abstract

Nitric oxide (NO) traffic within the reduced ferrousnitrosyl complex of endothelial nitric-oxide synthase (eNOS) has been studied by ultrafast time-resolved absorption spectroscopy. In the presence of tetrahydrobiopterin, the rate of NO rebinding to the heme upon photodissociation depends on the NO concentration. The time scale of this process, picoseconds to nanoseconds, precludes a diffusion from the solution toward the protein medium, and altogether the data point at a new NO binding site within the protein. Comparison of the kinetics of pterin-bound and -depleted eNOS points out that the existence of this new site depends on the presence of tetrahydrobiopterin. The new non-heme site may act as a "doorstep" to the heme pocket and control NO escape from eNOS. Nitric oxide (NO)1 is produced from L-arginine in the vascular endothelium by the endothelial isoform of nitric-oxide synthase, eNOS. NO production is crucial in the control of vascular tone, arterial pressure, smooth muscle cell proliferation, and platelet adhesion to the endothelial surface. NOS catalyzes the formation of NO and L-citrulline from L-arginine in the presence of oxygen and NADPH (1). The active protein is a homodimer (2). Upon Ca 2ϩ /calmodulin binding, the FAD of the reductase domain transfers reducing equivalents from NADPH to FMN, which then reduces the heme iron located in the oxygenase domain. This domain also includes binding sites for the substrate, L-arginine, and the cofactor, tetrahydrobiopterin (BH 4 ). The cofactor BH 4 is required for full activity of all NOS isoforms. Reconstitution of holoenzyme activity and BH 4 binding requires the presence of glutathione, necessary to reduce an oxidized cysteine (3). BH 4 binds at the edge of, and perpendicular to, the heme plane through an extensive network of hydrogen bonds (4 -6).In this report, we focus on the effect of NO concentration on the dynamics of NO released from the ferrous-nitrosyl complex, Fe(II)-NO (7), and its potential role in controlling NO release from NOS. The ultrafast rebinding of a dissociated NO molecule to the eNOS heme site, a process called geminate recombination, involves NO molecules that do not escape into the solvent. The kinetics of this rebinding are very sensitive to the environment of the heme. This ligand rebinding is studied at various NO concentrations.In this work, we show that varying the NO to the protein ratio induces changes in the geminate recombination rate for eNOS possessing bound BH 4 . In contrast, the rate of NO rebinding to BH 4 -depleted eNOS was independent of NO concentration and much slower than in the presence of the cofactor. Our data strongly suggest the existence of a new non-heme binding site for NO that depends on the presence of BH 4 . EXPERIMENTAL PROCEDURESMaterials-L-Arginine, ␤-NADPH (99.8% purity), (6R)5,6,7,8-tetrahydrobiopterin (BH 4 ), and calmodulin, as well as other chemicals used for buffer preparations, were purchased from Sigma. Diluted NO gas at 1 and 10% (v/v) in nitrogen and argon, respectively, and high purit...

Hydrophobic Distal Pocket Affects NO−Heme Geminate Recombination Dynamics in Dehaloperoxidase and H64V Myoglobin

“…The dependence of NO recombination on glycerol, temperature, and distal pocket mutation in myoglobin has been studied by a number of groups. ,− ,− ,− Time-resolved NO rebinding studies provide a general probe of distal pocket structure in heme proteins. ,− Recombination of NO is non-single-exponential in nearly every heme protein studied. In a previous study of NO recombination in HHMb, a maximum entropy model was applied to the NO rebinding kinetics in the presence of varying glycerol concentrations .…”

How does a valine residue that modulates heme-NO binding kinetics in inducible NO synthase regulate enzyme catalysis?

“…However, NOS activity is also influenced in a fundamental way by a heme-NO binding event that is an intrinsic feature of their catalysis [16][17][18][19][20]. Each newly-generated NO molecule has a high probability of binding to the NOS heme multiple times before it escapes from the heme pocket [21,22], and even NO released from the enzyme can rebind to the NOS heme if sufficient external concentrations are achieved (low lM NO) [19,22]. Accordingly, NOS heme-NO complexes can build up both in steady-state reactions and as a transient species during NOHA oxidation reactions catalyzed by NOS oxygenase domains (NOSoxy) under single turnover conditions [16][17][18][19].…”

Dynamic Regulation of the Inducible Nitric-oxide Synthase by NO

“…The effect of BH 4 on the rate of NO rebinding to ferric iNOSoxy contrasts the lack of such dependence in the case of the ferric eNOS (31). It is opposite to the one observed on NO rebinding to the ferrous eNOS (32). The simulations show that NO (represented by green and magenta bars) remains localized on the heme over 10 ps independently of BH 4 (Fig.…”