Formation of the reactive iron-oxo intermediate during the biosynthesis of nitric oxide by neuronal nitric oxide synthase and its possible mechanism in the generation of secondary free radicals
dc.contributor.author | Porasuphatana, Supatra | |
dc.date.accessioned | 2012-04-02T19:50:01Z | |
dc.date.available | 2012-04-02T19:50:01Z | |
dc.date.issued | 2001 | |
dc.identifier.uri | http://hdl.handle.net/10713/1212 | |
dc.description | University of Maryland, Baltimore. Pharmaceutical Sciences. Ph.D. 2001 | en_US |
dc.description.abstract | Nitric oxide synthases (NOSs) are hemoproteins that catalyze the formation of nitric oxide (NO•) and L-citrulline from L-arginine by a five-electron oxidation reaction. All NOS isoforms are in the same superfamily and share structural similarities with cytochrome P-450. The generation of free radicals by NOS is primarily regulated by the binding of L-arginine. During the oxidation of L-arginine to generate NO•, the transfer of electrons from reductase to oxygenase domain of NOS leads to changes in the oxidation state of heme iron, resulting in the formation of the perferryl iron-oxo intermediate of NOS (NOS-[Fe5+=O]3+). This dissertation aims to explore the possible role of NOS-[Fe5+=O]3+ in the generation of secondary free radicals. The NOS-[Fe5+=O] 3+, which formed only when L-arginine bound to the enzyme, was proven to abstract a hydrogen atom from substrates to generate carbon-centered free radicals. It was demonstrated that the abstraction of hydrogen atom by NOS-[Fe 5+=O]3+ occurred at a carbon alpha to a heteroatom, similar to hydroxylation reaction catalyzed by cytochrome P-450. Further investigation using potassium hydrogen persulfate (KHSO5) confirmed the formation of NOS-[Fe5+=O]3+ which may be generated via the rearrangement of NOS-[Fe3+-O-O-SO 3-] following the transfer of oxygen from KHSO 5 to NOS I. The NOS-[Fe5+=O]3+ generated by KHSO5-catalyzed NOS I was shown to generate NO• from L-arginine and alpha-hydroxyethyl radical from ethanol, resembling the incidences found with the NADPH/O2 NOS as a source of substrate oxidation. Deuterium isotope effect, using unlabeled ethanol and ethyl 1,1-d2 alcohol, revealed the importance of the breakage of C1-H bond as a partial rate-limiting step for the formation of carbon-centered free radicals by NOS. Taken together, this research illustrates the contribution of L-arginine binding to the formation of NOS reactive iron-oxo complex intermediate and the role of the iron complex in the generation of carbon-centered free radicals by the hydrogen atom abstraction. | en_US |
dc.language.iso | en_US | en_US |
dc.subject | Health Sciences, Pharmacology | en_US |
dc.title | Formation of the reactive iron-oxo intermediate during the biosynthesis of nitric oxide by neuronal nitric oxide synthase and its possible mechanism in the generation of secondary free radicals | en_US |
dc.type | dissertation | en_US |
dc.contributor.advisor | Rosen, Gerald M., Ph.D., J.D. | |
dc.identifier.ispublished | Yes |
