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Characterization of neuronal nitric-oxide synthase reductase activity

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dc.contributor.advisor Schimerlik, Michael I.
dc.creator Wolthers, Kirsten R.
dc.date.accessioned 2012-08-21T16:37:25Z
dc.date.available 2012-08-21T16:37:25Z
dc.date.copyright 2001-04-24
dc.date.issued 2001-04-24
dc.identifier.uri http://hdl.handle.net/1957/32665
dc.description Graduation date: 2001 en_US
dc.description.abstract During catalysis the flavoprotein domain of neuronal nitric-oxide synthase (nNOS) shuttles NADPH-derived reducing equivalents from FAD to FMN and then to the P450-heme enabling heme-based oxygen activation and subsequent NO-synthesis. The binding of Ca²⁺-activated calmodulin (Ca²⁺-CaM) to nNOS alleviates inhibition of flavin mediated electron transfer within the diflavin domain, which is demonstrated by the increase in the rate of 2,6-dichioroindoiphenol (DCIP) reduction by 2 to 3 fold and that of cytochrome c³⁺ by 10 to 20 fold. To investigate the effect of the Ca²⁺-CaM on the nNOS reductase activity, the steady-state kinetics of basal and CaM-stimulated reduction of these two substrates was studied. Parallel initial velocity patterns indicated that both substrates are reduced in a ping-pong mechanism. Product and dead-end inhibition data with DCIP as the electron acceptor were consistent with a di iso ping-pong bi-bi mechanism. In contrast, product and dead-end inhibition studies with cytochrome c³⁺ as the second substrate were consistent with an iso (two-site) ping-pong mechanism. Ca²⁺-CaM did not alter the proposed kinetic mechanisms; however, it did effect to varying degrees the (k[subscript cat]/K[subscript]m) for the various substrates. The pH-dependence of basal and CaM-stimulated reduction of DCIP revealed that ionizable groups involved in the binding of substrates and catalysis are not altered by Ca²⁺-CaM. However, the activated cofactor does influence catalytic rate constants and/or ionizable groups involved in cytochrome c³⁺ reduction. nNOS was found to abstract the pro-R (A-side) hydrogen from NADPH. Primary deuterium isotope effects (NADP(D)) and solvent isotope effects (SKIE) suggests that of the two half reactions, the reductive half reaction involving NADPH oxidation limits the overall reaction rate, but that hydride transfer to FAD is not the slow step. A small value of [supercript D](V/K)[subscript NADPH] (1.2-1.6) suggests hydride transfer is not the rate-limiting step within the reductive half-reaction. Large solvent kinetic isotope effects (SKIE) were observed on (V/K)[subscript cytc] for basal and CaM stimulated reduction of cytochrome c³⁺ suggesting that proton uptake from the solvent limits the rate of the oxidative half-reaction. A small SKIE on V and (V/K)[subscript NADPH] indicates that proton uptake does not limit the overall reaction rate. Proton inventory analysis revealed multiple transition-state protons contributed to the observed SKIE. en_US
dc.language.iso en_US en_US
dc.subject.lcsh Nitric-oxide synthase en_US
dc.title Characterization of neuronal nitric-oxide synthase reductase activity en_US
dc.type Thesis/Dissertation en_US
dc.degree.name Doctor of Philosophy (Ph. D.) in Biochemistry and Biophysics en_US
dc.degree.level Doctoral en_US
dc.degree.discipline Science en_US
dc.degree.grantor Oregon State University en_US
dc.contributor.committeemember Anderson, Sonia
dc.contributor.committeemember Mosbaugh, Dale
dc.contributor.committeemember Giovannoni, Stephen
dc.contributor.committeemember Wong, Isaac
dc.description.digitization File scanned at 300 ppi (Monochrome, 256 Grayscale) using Capture Perfect 3.0 on a Canon DR-9050C in PDF format. CVista PdfCompressor 4.0 was used for pdf compression and textual OCR. en_US
dc.description.peerreview no en_us

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