Structural and Functional Characterization of a Cytochrome P450 2B4 F429H Mutant with an Axial Thiolate鈥揌istidine Hydrogen Bond
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- 作者：Yuting Yang ; Haoming Zhang ; Dandamudi Usharani ; Weishu Bu ; Sangchoul Im ; Michael Tarasev ; Freeborn Rwere ; Naw May Pearl ; Jennifer Meagher ; Cuthbert Sun ; Jeanne Stuckey ; Sason Shaik ; Lucy Waskell
- 刊名：Biochemistry
- 出版年：2014
- 出版时间：August 12, 2014
- 年：2014
- 卷：53
- 期：31
- 页码：5080-5091
- 全文大小：430K
- ISSN：1520-4995
文摘
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 未N atom of His429 forms an H-bond with the axial Cys436 thiolate whereas the 蔚N 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鈥搕hiolate interaction demonstrates a significant electron withdrawing effect on the heme iron. Comparisons of structures of class I鈥揑V 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.