Dataset Information

Kinetics of CO Recombination to the Heme in Geobacillus Stearothermophilus Nitric Oxide Synthase.

ABSTRACT: We report the kinetics of CO rebinding to the heme in His134Ser, Ile223Val and His134Ser/Ile223Ser mutants of Geobacillus stearothermophilus nitric oxide synthase (gsNOS). The amplitudes of the two observed kinetics phases, which are insensitive to CO concentration, depend on enzyme concentration. We suggest that two forms of gsNOS are in equilibrium under the conditions employed (6.1-27 µM gsNOS with 20 or 100% CO atmosphere). The kinetics of CO rebinding to the heme do not depend on the identity of the NO-gate residues at positions 134 and 223.

SUBMITTER: Whited CA

PROVIDER: S-EPMC3747573 | biostudies-literature | 2013 Jul

REPOSITORIES: biostudies-literature

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Kinetics of CO Recombination to the Heme in <i>Geobacillus Stearothermophilus</i> Nitric Oxide Synthase.

Whited Charlotte A CA Warren Jeffrey J JJ Lavoie Katherine D KD Winkler Jay R JR Gray Harry B HB

Polyhedron 20130701

We report the kinetics of CO rebinding to the heme in His134Ser, Ile223Val and His134Ser/Ile223Ser mutants of <i>Geobacillus stearothermophilus</i> nitric oxide synthase (gsNOS). The amplitudes of the two observed kinetics phases, which are insensitive to CO concentration, depend on enzyme concentration. We suggest that two forms of gsNOS are in equilibrium under the conditions employed (6.1-27 µM gsNOS with 20 or 100% CO atmosphere). The kinetics of CO rebinding to the heme do not depend on the ...[more]

PMID: 23976816

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Project description:Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to L-citrulline and NO in a two-step process involving the intermediate N(?)-hydroxy-L-arginine (NHA). It was shown that Cpd I is the oxygenating species for L-arginine; the hydroperoxo ferric intermediate is the reactive intermediate with NHA. Methylation of the N(?)-OH and N(?)-H of NHA significantly inhibits the conversion of NHA into NO and L-citrulline by mammalian NOS. Kinetic studies now show that N(?)-methylation of NHA has a qualitatively similar effect on H?O?-dependent catalysis by bacterial gsNOS. To elucidate the effect of methylating N(?)-hydroxy L-arginine on the properties and reactivity of the one-electron-reduced oxy-heme center of NOS, we have applied cryoreduction/annealing/EPR/ENDOR techniques. Measurements of solvent kinetic isotope effects during 160 K cryoannealing cryoreduced oxy-gsNOS/NHA confirm the hydroperoxo ferric intermediate as the catalytically active species of step two. Product analysis for cryoreduced samples with methylated NHA's, NHMA, NMOA, and NMMA, annealed to 273 K, show a correlation of yields of L-citrulline with the intensity of the g 2.26 EPR signal of the peroxo ferric species trapped at 77 K, which converts to the reactive hydroperoxo ferric state. There is also a correlation between the yield of L-citrulline in these experiments and k(obs) for the H?O?-dependent conversion of the substrates by gsNOS. Correspondingly, no detectable amount of cyanoornithine, formed when Cpd I is the reactive species, was found in the samples. Methylation of the NHA guanidinium N(?)-OH and N(?)-H inhibits the second NO-producing reaction by favoring protonation of the ferric-peroxo to form unreactive conformers of the ferric-hydroperoxo state. It is suggested that this is caused by modification of the distal-pocket hydrogen-bonding network of oxy gsNOS and introduction of an ordered water molecule that facilitates delivery of the proton(s) to the one-electron-reduced oxy-heme moiety. These results illustrate how variations in the properties of the substrate can modulate the reactivity of a monooxygenase.

Dataset Information

Kinetics of CO Recombination to the Heme in Geobacillus Stearothermophilus Nitric Oxide Synthase.

Publications

Kinetics of CO Recombination to the Heme in <i>Geobacillus Stearothermophilus</i> Nitric Oxide Synthase.

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