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Title: Electron transfer within the reductase domain of nueronal nitric oxide synthase
Author: Knight, Kirsty
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 2003
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The reductase domain of neuronal nitric oxide synthase (nNOS) was purified in the presence and absence of calmodulin. In the absence of calmodulin (CaM), the majority of the protein was isolated in a proteolysed form. The addition of EDTA produced the CaM-free form of the protein. Static fluorescence and N-terminal sequence analysis confirmed the integrity of the CaM-binding domain in the CaM-bound form of the protein. The steady-state kinetics of the reductase domain showed the expected ten-fold stimulation in the rate of cytochrome c reduction in the presence of CaM. The reduction of the FAD and FMN cofactors by NADPH was studied by anaerobic stopped-flow spectroscopy. Photodiode array, single wavelength absorption and fluorescence detection were used to develop a new kinetic scheme for electron transfer in the reductase domain of the nNOS consisting of five spectral intermediates. The kinetics of the CaM-free form of the reductase domain do not differ significantly from those of the CaM-bound form. The lack of formation of a blue semiquinone under stopped-flow conditions indicated that electron transfer in the reductase domain is gated, probably by NADP+ release. NADP+ was found to slow the first step in the catalytic scheme of the enzyme, namely the formation of an equilibrium between an NADPH-enzyme charge-transfer species and two-electron-reduced enzyme bound to NADP+. Preliminary studies on the inhibition of the kinetics of the reductase domain of nNOS by fragments of NADPH identified which parts of the cofactor interact with the enzyme. The NMN portion of NADPH did not inhibit CaM-bound nNOS. The strongest inhibition was observed with the 2'-AMP portion of the cofactor. These studies constitute a detailed study of the kinetics of electron transfer in the reductase domain of the nNOS in the presence and absence of CaM.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available