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 (H(4)Bip) are not fully understood. Here we have investigated possible allosteric and stabilizing effects of H(4)Bip 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 H(4)Bip activated recombinant human NOS-I to increase L-arginine turnover. Consistent with this was the observation that H(4)Bip, but not the pterin-based NOS inhibitor 2-amino-4,6-dioxo-3,4,5,6,8,8a,9,10-octahydrooxazolo[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 H(4)Bip binding to pig brain NOS-I. Secondly, we investigated the stabilization of NOS quaternary structure by H(4)Bip in relation to uncoupled catalysis. Under catalytic assay conditions and in the absence of H(4)Bip, 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 N(omega)-nitro-L-arginine. Importantly, H(4)Bip was found to react chemically with superoxide (O(2)(-.)) and enzyme-bound H(4)Bip was consumed under O(2)(-.)-generating conditions in the absence of substrate. These results suggest that H(4)Bip allosterically activates NOS-I and stabilizes quaternary structure by a novel mechanism involving the direct interception of auto-damaging O(2)(-.).

Nitric Oxide Synthase Dysfunction Contributes to Impaired Cerebroarteriolar Reactivity in Experimental Cerebral Malaria

“…The enhancement of oxidative stress as a result of NOS uncoupling could exacerbate NOS dysfunction by destabilizing the quaternary structure of the NOS dimers, causing them to dissociate into their nonfunctional monomeric forms [31], [32]. In the present study, increased levels of eNOS and nNOS monomerization indicate that enhanced conformational inactivation of eNOS and nNOS under conditions of elevated oxidation stress in the brain could contribute to NOS dysfunction in ECM.…”

“…Functional NOS dimers can become unstable and dissociate into their nonfunctional monomeric forms under conditions of enhanced oxidative stress [31], [32]. We asked if enhanced conformational inactivation of NOS isoforms by monomerization could contribute to NOS dysfunction in the brain during ECM development.…”

Bi-modal dose-dependent cardiac response to tetrahydrobiopterin in pressure-overload induced hypertrophy and heart failure

“…Decreased BH4 bioavailability leads to conformational changes in the NOS homodimer, leading to the phenomenon known as NOS-uncoupling, where less NO is generated and instead the enzyme produces superoxide [6]. Studies in the heart and vasculature have shown BH4 deficiency or its oxidation to BH2 increases NOS-derived reactive oxygen species (ROS) generation.…”

Renal Response to L-Arginine in Diabetic Rats. A Possible Link between Nitric Oxide System and Aquaporin-2

“…So, it may be that L-Arg supplementation enhances NOS activity decreasing ROS formation. This may explain the increase in NOS activity in the diabetic rats, but not in the control rats, which led us to think that L-Arg is more probably acting as an allosteric activator of the enzyme in both control and diabetic rats [45].…”