title: Structural and functional characterization of a cytochrome P450 2B4 F429H mutant with an axial thiolate-histidine hydrogen bond.
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.
authors.affiliation: Department of Anesthesiology, University of Michigan and VA Medical Center , 2215 Fuller Road, Building 31, Room 225, Ann Arbor, Michigan 48105, United States.
authors.forename: Yuting
authors.initials: Y
authors.lastname: Yang
authors.forename: Haoming
authors.initials: H
authors.lastname: Zhang
authors.forename: Dandamudi
authors.initials: D
authors.lastname: Usharani
authors.forename: Weishu
authors.initials: W
authors.lastname: Bu
authors.forename: Sangchoul
authors.initials: S
authors.lastname: Im
authors.forename: Michael
authors.initials: M
authors.lastname: Tarasev
authors.forename: Freeborn
authors.initials: F
authors.lastname: Rwere
authors.forename: Naw May
authors.initials: NM
authors.lastname: Pearl
authors.forename: Jennifer
authors.initials: J
authors.lastname: Meagher
authors.forename: Cuthbert
authors.initials: C
authors.lastname: Sun
authors.forename: Jeanne
authors.initials: J
authors.lastname: Stuckey
authors.forename: Sason
authors.initials: S
authors.lastname: Shaik
authors.forename: Lucy
authors.initials: L
authors.lastname: Waskell
mesh_terms: D019943:Amino Acid Substitution; D001189:Aryl Hydrocarbon Hydroxylases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D018360:Crystallography, X-Ray; D000072467:Cytochrome P450 Family 2; D015786:Cytochromes b5 / Q000378:metabolism; D004579:Electron Transport; D006418:Heme / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D006860:Hydrogen Bonding; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D010084:Oxidation-Reduction; D010649:Phenylalanine / Q000737:chemistry; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D013816:Thermodynamics
chemical_list: D050505:Mutant Proteins; D011994:Recombinant Proteins; D006418:Heme; D010649:Phenylalanine; D006639:Histidine; D015786:Cytochromes b5; D001189:Aryl Hydrocarbon Hydroxylases; D000072467:Cytochrome P450 Family 2; C465241:cytochrome P-450 CYP2B4 (rabbit); D009251:NADPH-Ferrihemoprotein Reductase