Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.422868
Title: Characterisacion of flavocytochrome P450 BM3 site directed mutants with novel heme ligation state
Author: Girvan, Hazel M.
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 2005
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Abstract:
Uniquely among cytochromes P450, family 4 P450s have a conserved glutamate covalently attaching heme to the protein backbone. In the related Bacillus megaterium P450 BM3, the corresponding residue is alanine (A264). The relevant mutation (A264E) was constructed and characterised spectroscopically, kinetically and structurally. Heme is not covalently ligated in A264E BM3. Instead, E264 coordinates directly to heme iron, generating novel Glu/Cys heme axial ligation. E264 coordination is promoted by fatty acid substrate binding. Both substrate-free and palmitoleate-bound A264E structures were solved and showed that one of two molecules in the substrate-free asymmetric unit had E264 ligation to heme iron, whilst the other had distal water. In all crystal forms, the structure was in a conformation only previously observed in substrate-bound wild-type enzyme. BM3 exists in a conformational equilibrium, and fatty acids bind preferentially to the "substrate-bound" conformation rather than substrate inducing conformational change per se. in light of A264E's novel heme axial ligation state, other A264 mutants were generated. A264H and A264K mutants showed His/Cys and His/Lys ligation. Both mutants showed completely coordinated by the amino acid sidechains in presence or absence of substrates, and were inactive. A264Q and A264M mutants had limited catalytic activity and partial Gln/Cys or Met/Cys ligation, whilst A264C also showed limited activity, and partial Cys/Cys ligation from spectroscopic studies (but none detected in the crystal). Atomic structures were solved for all mutants and spectroscopic analysis provided first characterisation of novel heme iron ligand sets.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.422868  DOI: Not available
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