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Structural and functional characterization of a cytochrome P450 2B4 F429H mutant with an axial thiolate-histidine hydrogen bond.
- Source :
-
Biochemistry [Biochemistry] 2014 Aug 12; Vol. 53 (31), pp. 5080-91. Date of Electronic Publication: 2014 Jul 31. - Publication Year :
- 2014
-
Abstract
- The structural basis of the regulation of microsomal cytochrome P450 (P450) activity was investigated by mutating the highly conserved heme binding motif residue, Phe429, on the proximal side of cytochrome P450 2B4 to a histidine. Spectroscopic, pre-steady-state and steady-state kinetic, thermodynamic, theoretical, and structural studies of the mutant demonstrate that formation of an H-bond between His429 and the unbonded electron pair of the Cys436 axial thiolate significantly alters the properties of the enzyme. The mutant lost >90% of its activity; its redox potential was increased by 87 mV, and the half-life of the oxyferrous mutant was increased ∼37-fold. Single-crystal electronic absorption and resonance Raman spectroscopy demonstrated that the mutant was reduced by a small dose of X-ray photons. The structure revealed that the δN atom of His429 forms an H-bond with the axial Cys436 thiolate whereas the εN atom forms an H-bond with the solvent and the side chain of Gln357. The amide of Gly438 forms the only other H-bond to the tetrahedral thiolate. Theoretical quantification of the histidine-thiolate interaction demonstrates a significant electron withdrawing effect on the heme iron. Comparisons of structures of class I-IV P450s demonstrate that either a phenylalanine or tryptophan is often found at the location corresponding to Phe429. Depending on the structure of the distal pocket heme, the residue at this location may or may not regulate the thermodynamic properties of the P450. Regardless, this residue appears to protect the thiolate from solvent, oxidation, protonations, and other deleterious reactions.
- Subjects :
- Amino Acid Substitution
Aryl Hydrocarbon Hydroxylases genetics
Binding Sites genetics
Crystallography, X-Ray
Cytochrome P450 Family 2
Cytochromes b5 metabolism
Electron Transport
Heme chemistry
Histidine chemistry
Hydrogen Bonding
Kinetics
Models, Molecular
Mutagenesis, Site-Directed
Mutant Proteins chemistry
Mutant Proteins genetics
Mutant Proteins metabolism
NADPH-Ferrihemoprotein Reductase metabolism
Oxidation-Reduction
Phenylalanine chemistry
Protein Conformation
Recombinant Proteins chemistry
Recombinant Proteins genetics
Recombinant Proteins metabolism
Structural Homology, Protein
Substrate Specificity
Thermodynamics
Aryl Hydrocarbon Hydroxylases chemistry
Aryl Hydrocarbon Hydroxylases metabolism
Subjects
Details
- Language :
- English
- ISSN :
- 1520-4995
- Volume :
- 53
- Issue :
- 31
- Database :
- MEDLINE
- Journal :
- Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 25029089
- Full Text :
- https://doi.org/10.1021/bi5003794