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Title: Structural and functional studies of nitric oxide synthase
Author: Stevenson, Thirza Helen
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 2001
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The oxygenase domains of rat brain nNOS (amino acids 221-724) and murine macrophage iNOS (residues 66-498) have been expressed and purified from Escherichia coli (E. coli). The two haem domain proteins have been characterised with respect to substrate and cofactor binding. The nNOS protein could not be purified to a high degree and bound less haem and BH4 than expected. The purified iNOS haem domain, however, contained its full complement of iron and zinc, had an optimal subunit:haem: BH4 stoichiometry of 1:1:1 and bound L-arginine with a similar affinity as the E. coli-generated full-length murine iNOS. Optical perturbation spectroscopy and NMR paramagnetic relaxation experiments were used to study the interaction between NOS substrates, or inhibitors, and the reactive centre of the murine iNOS haem domain. Type I ligands, such as the substrates L-arginine bound near the haem and converted any hexa-co-ordinate low-spin haem iron to a penta-co-ordinate high-spin form. Type II ligands, such as imidazole, however, co-ordinated directly to the iNOS haem iron resulting in enzyme with a six-co-ordinate low-spin haem iron. To obtain information on the orientation of ligands within the iNOS active site, the NMR experiments required ligands to be weakly binding and in fast-exchange on the NMR timescale with the iNOS haem domain. Although spectral titrations identified ligands with near millimolar binding affinities, NMR experiments indicated that the ligands did not satisfy the fast-exchange criteria. Laser flash photolysis and stopped-flow spectroscopy were used to examine the kinetics of ligand binding to the iNOS haem domain. Studies of Type I ligand binding to the iNOS haem domain-CO complex supported the view that the haem domain exists in different conformational states, corresponding to 'open' and 'closed' forms of the ligand access channel. In the absence of a Type I ligand and BH4, the iNOS haem domain appeared to have a relatively open distal pocket that allowed CO to bind unhindered. In the presence of BH4 and Type I ligand, however, the enzyme adopted a more closed structure that greatly reduced ligand access to the haem iron.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available