Structural and spectroscopic analysis of the F393H mutant of flavocytochrome p450 BM3

被引:47
作者
Ost, TWB
Munro, AW
Mowat, CG
Taylor, PR
Pesseguiero, A
Fulco, AJ
Cho, AK
Cheesman, MA
Walkinshaw, MD
Chapman, SK
机构
[1] Univ Edinburgh, Dept Chem, Edinburgh EH9 3JJ, Midlothian, Scotland
[2] Univ Leicester, Dept Biochem, Leicester LE1 7RH, Leics, England
[3] Univ Edinburgh, ICMB, Edinburgh EH9 3JR, Midlothian, Scotland
[4] Univ Calif Los Angeles, Sch Med, Dept Biol Chem, Los Angeles, CA 90095 USA
[5] Univ Calif Los Angeles, Sch Med, Dept Mol & Med Pharmacol, Los Angeles, CA 90095 USA
[6] Univ E Anglia, Sch Chem Sci, Norwich NR4 7TJ, Norfolk, England
关键词
D O I
10.1021/bi010717e
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 Angstrom 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 nn 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.
引用
收藏
页码:13430 / 13438
页数:9
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