In the preceding paper in this issue [Ost, T. W. B., Miles, C. S., Munro, A. W., Murdoch, J., Reid, G. A., and Chapman, S. K. (2001) Biochemistry 40, 13421-13429], we have established that the primary role of the phylogenetically conserved phenylalanine in flavocytochrome P450 BM3 (F393) is to control the thermodynamic properties of the heme iron, so as to optimize electron-transfer both to the iron (from the flavin redox partner) and onto molecular oxygen. In this paper, we report a detailed study of the F393H mutant enzyme, designed to probe the structural, spectroscopic, and metabolic profile of the enzyme in an attempt to identify the factors responsible for causing the changes. The heme domain structure of the F393H mutant has been solved to 2.0 A resolution and demonstrates that the histidine replaces the phenylalanine in almost exactly the same conformation. A solvent water molecule is hydrogen bonded to the histidine, but there appears to be little other gross alteration in the environment of the heme. The F393H mutant displays an identical ferric EPR spectrum to wild-type, implying that the degree of splitting of the iron d orbitals is unaffected by the substitution, however, the overall energy of the d-orbitals have changed relative to each other. Magnetic CD studies show that the near-IR transition, diagnostic of heme ligation state, is red-shifted by 40 nm in F393H relative to wild-type P450 BM3, probably reflecting alteration in the strength of the iron-cysteinate bond. Studies of the catalytic turnover of fatty acid (myristate) confirms NADPH oxidation is tightly coupled to fatty acid oxidation in F393H, with a product profile very similar to wild-type. The results indicate that gross conformational changes do not account for the perturbations in the electronic features of the P450 BM3 heme system and that the structural environment on the proximal side of the P450 heme must be conformationally conserved in order to optimize catalytic function.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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