The first part of this thesis focused on methodological developments of single- crystal resonance Raman spectroscopy (SCRR) and its novel combination with X-ray crystallography to assign and validate redox and ligand states. The utility of both methods was initially tested on CYTcp from Alcaligenes xylosoxidans (AXCP) crystals as it has been well characterised in solution including resonance Raman spectroscopy and is represented in chapter 4. The novel approach has been used for the spectroscopic validation of all crystal structures presented in this thesis. AXCP shares a similar heme environment with CYTcp from Rhodopseudomonas palustris (RPCP), which exists entirely as a monomer. Research has indicated that the unusual'distal-to-proximal' NO conversion in AXCP is highly dependent on the proximal Arg124 and distal Leu16 residue. The effect of the distal Leu residue in AXCP and RPCP has been explored in chapter 5 and 6 by site-directed mutants to make more room at the distal site and lift steric hindrance. The ligand-bound CYTcp were structurally, kinetically and spectroscopically characterised. The L 16VAXCP, L 161AXCP and L 121RPCP mutants showed a dramatic boost in NO affinity of the distal 6c-NO complex and hindering the conversion to the proximal 5c-NO. Both AXCP mutants displayed a -30-fold faster kon(NO) and -10-2-fold slower koff(NO) indicating that the distal residue modulates the balance of affinities of both heme sites. In contrast, the high distal koff(NO) in AXCP supports a fast distal NO release compared to the proximal NO in the
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:701798 |
| Date | January 2015 |
| Creators | Kekilli, Demet |
| Publisher | University of Essex |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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