Gating NO Release from Nitric Oxide Synthase

Abstract

We have investigated the kinetics of NO escape from Geobacillus stearothermophilus nitric oxide synthase (gsNOS). Previous work has indicated that NO release was gated at position 223 in mammalian enzymes; our kinetics experiments include mutants at that position along with measurements on the wild type enzyme. Employing stopped flow UV-vis methods, reactions were triggered by mixing reduced enzyme/N-hydroxy-L-arginine complex with aerated buffer solution. NO release kinetics were obtained for wt NOS and three mutants (H134S, I223V, H134S/I223V). We have confirmed that wt gsNOS has the lowest NO release rate of known NOS enzymes, whether bacterial or mammalian. We also have found that steric clashes at positions 223 and 134 hinder NO escape, as judged by enhanced rates in the single mutants. The empirical rate of NO release from the gsNOS double mutant (H134/I223V) is nearly as rapid as that of the fastest mammalian enzymes, demonstrating that both positions 223 and 134 function as gates for escape of the product diatomic molecule.

Kinetics of CO recombination to the heme in Geobacillus stearothermophilus nitric oxide synthase

“…Our results for CO-rebinding to the heme in mutants of gsNOS contrast with those for NO-release in single-turnover experiments [14]. In the NO-release study we demonstrated the importance of NO-gates at positions 134 and 223.…”

“…Interconversion of Fe A and Fe B must be slower than CO-rebinding to account for the observed kinetics. We note that residues at gsNOS NO-gate positions 134 and 223 only slightly affect the amplitudes in Table 1, in contrast to results from single-turnover NO release experiments [14]. Biphasic kinetics also were observed for CO-rebinding in mouse inducible NOS [36] and neuronal NOS [37].…”

“…The 200-fold difference in NO-release rates between bacterial and mammalian enzymes [14] is striking, particularly in view of high sequence similarities and conserved folds among NOS isoforms. Crystallographic studies suggest that Val is the first gate for NO release in mammalian enzymes (e.g.…”

“…We have investigated four site-directed mutants of gsNOS that have different amino acids at the two NO-gate positions, 134 and 223 (Figure 1): wild type (wt), I223V, H134S, and the double mutant H134S/I223V. In single-turnover experiments we found that the rate of NO release in the gsNOS double mutant (1.0 s −1 ) approached that of fast NO releasing isoforms at 298 K [14]. We have extended our work on these isoforms to include kinetics of CO rebinding to the gsNOS heme.…”

The Initial Common Pathway of Inflammation, Disease, and Sudden Death

“…Exogenous surfactant-induced interfacial surface tension, i.e., "water stress," is the biophysical basis [1][2][3] for the generalized Sanarelli-Shwartzman phenomenon, generalized thrombohemorrhagic phenomena, the nonspecific generalized stress adaptation syndrome, the systemic inflammatory response syndrome, immune cell activation, macropinocytosis [73,[304][305][306], vesiculation [77], adhesion [63,64,78], engulfment [45,46,256], envelopment [309], fusion [74,77,78,172,[309][310][311], podokinesis [312][313][314][315], transcellular diapedesis [316] and nutritive endocytosis [317][318][319][320]. These highly-stereotyped supramolecular (mesoscopic), quantum coherent, biological processes are examples of the biophysical properties which motivate homeopathy.…”