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| | {{STRUCTURE_1jme| PDB=1jme | SCENE= }} | | {{STRUCTURE_1jme| PDB=1jme | SCENE= }} |
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| - | '''Crystal Structure of Phe393His Cytochrome P450 BM3'''
| + | ===Crystal Structure of Phe393His Cytochrome P450 BM3=== |
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| - | ==Overview==
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| - | 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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| | ==About this Structure== | | ==About this Structure== |
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| | [[Category: Fatty acid hydroxylase]] | | [[Category: Fatty acid hydroxylase]] |
| | [[Category: Monooxygenase]] | | [[Category: Monooxygenase]] |
| - | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Fri May 2 21:24:24 2008'' | + | |
| | + | ''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Tue Jul 1 20:28:00 2008'' |
Revision as of 17:28, 1 July 2008
Template:STRUCTURE 1jme
Crystal Structure of Phe393His Cytochrome P450 BM3
Template:ABSTRACT PUBMED 11695889
About this Structure
1JME is a Single protein structure of sequence from Bacillus megaterium. Full crystallographic information is available from OCA.
Reference
Structural and spectroscopic analysis of the F393H mutant of flavocytochrome P450 BM3., Ost TW, Munro AW, Mowat CG, Taylor PR, Pesseguiero A, Fulco AJ, Cho AK, Cheesman MA, Walkinshaw MD, Chapman SK, Biochemistry. 2001 Nov 13;40(45):13430-8. PMID:11695889
Page seeded by OCA on Tue Jul 1 20:28:00 2008
