Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.651338
Title: Calmodulin activation of the reductase domain of mammalian neuronal nitric oxide synthase
Author: Garnaud, Pierre-Emmanuel F.
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2006
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Abstract:
In order to investigate the CaM activation mechanism of nNOS, the effect of the conformational changes of nNOSrd and the binding of NADP(H) on the redox potential of the flavins, cofactors were assessed for the isolated FAD and FMN sub-domains by OTTLE potentiometry. The results showed that the presence of the FAD/FMN sub-domain interface does not alter the thermodynamic properties of the redox couples involved in the catalysis. This is consistent with the fact that CaM binding has a small effect on the flavins reduction potentials. Only the FMN/FMNH· redox couple was found to be stabilised by the presence of the FAD sub-domain (increase of 80 mV). The same redox couple was also kinetically stabilised toward oxidation. The isolated FAD sub-domain was found to have similar redox potentials to the isolated nNOSrd. In the presence of NADP+, both the FAD sub-domain and the nNOSrd formed the charge-transfer complex with a long-wavelength absorption band centred at 780nm. Formation of this complex was found to stabilise the FADH·/FADH- redox couple by approx 30 mV. It is possible that in the CaM-free enzyme, the conformation of the bound NADP+ may control both electron transfers between the FAD and FMN and from FMN to heme by modulating the potential of the FAD hydroquinone. The accessibility of the FMN cofactor to the heme was assessed. The results showed that if the FMN, in the isolated FMN domain, is assumed to be fully accessible, then it is 100% accessible in the CaM-bound enzyme, 45% accessible in the uncompleted enzyme and only 3% accessible in the NADPH-bound nNOSrd in the absence of CaM. This suggests that the binding of CaM is responsible for a structural reorganisation of nNOS rd that “unlocks” the conformation of the enzyme and enables the FMN sub-domain motion in order to shuttle an electron from FAD cofactor to the heme. The specificity of NADP(H) in repressing the electron transfer from the reductase domain to cytochrome c was studied by using NADP(H) analogues. Results showed that the specificity of NADPH in inducing nNOS rd conformational change relies upon the interaction of both the tightly-bound ADP substituents and the labile nicotinamide substituents and that the tightly bound ADP substituents is essential to position the nicotinamide moieties for full electron transfer repression. It appears that the “locked” conformation of the enzyme, believed to inhibit the electron transfer to the heme, is specific for the NADP(H).
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.651338  DOI: Not available
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