Allosteric regulation of neuronal nitric oxide synthase by tetrahydrobiopterin and suppression of auto-damaging superoxide

Abstract

The underlying mechanisms regulating the activity of the family of homodimeric nitric oxide synthases (NOSs) and, in particular, the requirement for (6R)-5,6,7,8-tetrahydro-L-biopterin (H4Bip) are not fully understood. Here we have investigated possible allosteric and stabilizing effects of H4Bip on neuronal NOS (NOS-I) during the conversion of substrate, L-arginine, into L-citrulline and nitric oxide. Indeed, in kinetic studies dual allosteric interactions between L-arginine and H4Bip activated recombinant human NOS-I to increase L-arginine turnover. Consistent with this was the observation that H4Bip, but not the pterin-based NOS inhibitor 2-amino-4,6-dioxo-3,4,5,6,8,8a,9,10-octahydro-oxazolo[1,2-f]-pteridine (PHS-32), caused an L-arginine-dependent increase in the haem Soret band, indicating an increase in substrate binding to recombinant human NOS-I. Conversely, L-arginine was observed to increase in a concentration-dependent manner H4Bip binding to pig brain NOS-I. Secondly, we investigated the stabilization of NOS quaternary structure by H4Bip in relation to uncoupled catalysis. Under catalytic assay conditions and in the absence of H4Bip, dimeric recombinant human NOS-I dissociated into inactive monomers. Monomerization was related to the uncoupling of reductive oxygen activation, because it was inhibited by both superoxide dismutase and the inhibitor NΩ-nitro-L-arginine. Importantly, H4Bip was found to react chemically with superoxide (O2-−) and enzyme-bound H4Bip was consumed under O2-−-generating conditions in the absence of substrate. These results suggest that H4Bip allosterically activates NOS-I and stabilizes quaternary structure by a novel mechanism involving the direct interception of auto-damaging O2-−.

Modulating endothelial nitric oxide synthase: a new cardiovascular therapeutic strategy

“…When the bioavailability of BH 4 or L-arginine declines, the eNOS dimer demonstrates a less tightly packed oxidase domain and a higher sensitivity to proteolysis (103). Multiple pleiotropic roles of BH 4 in eNOS enzymatic function have been demonstrated: BH 4 1) facilitates the binding of L-arginine substrate to eNOS, 2) shifts the heme iron into a high spin state that increases enzyme activity, 3) is involved in electron transfer, and 4) stabilizes the NOS dimer (24,63,65,108). Stoll et al (128) demonstrated that eNOS also stabilizes BH 4 by controlling its protonation state.…”

Tetrahydrobiopterin and Cardiovascular Disease

“…Vasoactive intestinal peptide oxidant effects and can scavenge NOS derived reactive nitrogen and oxygen species. 37,46 When BH 4 bioavailability declines, NOS undergoes multiple changes. The dimer architecture is altered possibly because of malrotation of the oxidase domains to yield "molecular" uncoupling, 47,48 and the catalytic activity becomes "functionally" uncoupled.…”

“…34 -36 NOS-bound BH 4 may act as a redox-active cofactor via an unknown mechanism. 34 BH 4 increases substrate affinity of NOS 21,35,37 and participates in the electron transfer process, being converted to BH 3 . radical during the NOS catalytic cycle and then restored to BH 4 .…”

Further evidence for the role of nitric oxide in the antiarrhythmic effect of ischaemic preconditioning: the effect of peroxynitrite and changes in NOS-dependent NO production

“…It is well known that NOS primary produces NO in the presence of adequate supply of substrate and cofactors, such as tetrahydrobiopterin (BH4), NADPH, FAD and flavin mononucleotide (Cosentino and Lüscher, 1999). Among these BH4 seems to be an essential cofactor for NOS to produce NO (Xia et al, 1998) by stabilizing NOS dimmers (reductase and oxygenase domains; Moens and Kass, 2006), increasing substrate affinity to the enzyme (Kotsonis et al, 2000) and inhibiting NOS-mediated superoxide production (Xia et al, 1998). There is also evidence that under ischaemic conditions the bioavailability of BH4 is reduced and this, rather than the inadequate substrate availability (Chatterjee and Catravas, 2008), results in uncoupling of NOS leading to superoxide rather than to NO production (Vasquez-Vivar et al, 1998).…”

“…For example, it has been shown that an excess in superoxide production would deactivate NOS, resulting in reduced free NO levels and subsequent peroxynitrite formation (Yaqoob et al, 1996). Similarly, under certain conditions the excessively generated NO can regulate, through a negative feedback (auto-inhibitory) mechanism the subsequent enzymatic synthesis of NO (Rengasamy and Johns 1993;Kotsonis et al, 1999Kotsonis et al, , 2000. As to whether in our experimental circumstances these radicals are, indeed, involved in the regulation of NOS is not known, what is certain that 5 min after the release of the second PC occlusion the activity of NOS has returned to the normal value, whereas the concentration of NO, superoxide and nitrotyrosine is remained to be still elevated.…”