Role of Tetrahydrobiopterin in Nitric Oxide Synthesis

The biosynthesis of nitric oxide (NO) is catalyzed by nitric-oxide synthase (NOS), which converts L-arginine into L-citrulline and NO in two distinct reactions with intermediate formation of N-hydroxy-L-arginine. Both reactions consume O2 and NADPH-derived electrons. NOS is a modular enzyme consisting of reductase and oxygenase domains. Catalysis takes place at a cytochrome P450-type heme in the oxygenase domain; the reductase domain contains FAD and FMN moieties that shuttle the required electrons from NADPH to the heme. NO synthesis is strictly dependent on the presence of tetrahydrobiopterin (BH4), which binds in the immediate vicinity of the heme. BH4 functions as an obligatory 1-electron donor in the reductive activation of the oxyferrous complex that is the last experimentally observed intermediate in the reaction cycle. BH4 may also be involved in the protonation of the reduced oxycomplex, and participation at later reaction steps is conceivable as well. Despite considerable progress in recent years, there are still many aspects concerning the role of BH4 that have not been studied in any detail yet. The present project aims to resolve some of the remaining questions. (i) Whereas BH4 reduces the oxyferrous complex that is formed after reduction of and oxygen binding to the heme, it does not react with the ferric enzyme. We will look into the reason for this and explore the redox potential of NOS-bound BH4. (ii) Before the reaction of NOS with the intermediate N-hydroxy-L-arginine can take place, the BH3 o radical that is formed in the reaction with Arg must be reduced by an electron ultimately originating from the reductase domain. However, particularly in the case of the endothelial isoform of NOS, there appears to be a discrepancy between the stability of the BH3 o radical and the slow rate of electron transfer from FMN to the heme. We will investigate the pathway of regeneration of BH4 and assess if the rate of interdomain electron transfer is sufficiently high to account for the rate of BH4 regeneration. (iii) Finally, we will explore the hypothesis that the FMN semiquinone, which has an oxidation potential precluding its participation in heme reduction, may function as an electron donor to the pterin radical under certain conditions. We will perform most of these studies with standard and stopped-flow/rapid-scan UV/visible spectroscopy. Additionally, we will apply more specialized techniques, partly in collaboration with other research groups, such as low-temperature optical spectroscopy, electron paramagnetic resonance spectroscopy, rapid-freeze and freeze-quench methods, and electrochemistry. We will carry out these studies with full-length NOS and/or the isolated oxygenase domains of both constitutive isoforms (endothelial and neuronal NOS).

Nitric oxide (NO) partakes in a wide array of physiological processes that include vasodilatation, smooth muscle relaxation, neurotransmission, and the immune response. The main source of nitric oxide in the body is the transformation of the amino acid L-arginine into L-citrulline and NO by the enzyme nitric-oxide synthase (NOS). This reaction critically depends on the presence of the cofactor tetrahydrobiopterin (BH4). In the absence of BH4, NOS cannot produce NO, but reduces oxygen (O2) to superoxide (O2) instead, with potentially devastating consequences. It has long been known that the binding of BH4 to NOS (2 equivalents per enzyme molecule) is not described by a simple equilibrium. We now discovered that, initially, NOS that is completely devoid of BH4 does bind BH4 in a simple reversible reaction. However, in a process that takes a few minutes to complete, one BH4 molecule is bound quasi-irreversibly, whereas the binding of the second molecule remains unaffected. An important consequence of this phenomenon is that NOS will never switch towards 100 % O2 production, but will still form O2 and NO simultaneously even when BH4 gets totally depleted or oxidized.S-Nitrosothiols are increasingly regarded as important signaling molecules in their own right in biology and therefore as potential pharmaceutical targets. However, controversy still surrounds the mechanism of their biological generation. We identified a novel reaction by which very low NO concentrations are able to efficiently nitrosate thiols. This reaction is strongly stimulated in the presence of physiological concentrations of magnesium cations. On the basis of kinetic studies of the nitrosation by NO of glutathione (GHS) and several other thiols we proposed a detailed mechanism for this reaction.In recent years hydrogen sulfide (H2S) has emerged as an important small signaling molecule in addition to NO. It has also become apparent that there are many points of interaction between the signaling pathways of NO and H2S, with both stimulatory and inhibitory effects. Our studies have demonstrated that H2S directly inhibits NO synthesis by NOS, but only at fairly high (104 M) concentrations and in a reaction that is reversible. However, we also found that the required concentration of H2S decrease (105 M) when NO is present as well and, importantly, in that case inhibition becomes irreversible. This phenomenon is also induced by NO originating from NOS itself. This inactivation may therefore offer a pathway for protective feedback inhibition in cases of pathophysiologically high NO/ H2S formation.