Structural and Functional Characterization of a Cytochrome P450 2B4 F429H Mutant with an Axial Thiolate–Histidine Hydrogen Bond
[Image: see text] 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 ki...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American
Chemical Society
2014
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4131899/ https://www.ncbi.nlm.nih.gov/pubmed/25029089 http://dx.doi.org/10.1021/bi5003794 |
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author | Yang, Yuting Zhang, Haoming Usharani, Dandamudi Bu, Weishu Im, Sangchoul Tarasev, Michael Rwere, Freeborn Pearl, Naw May Meagher, Jennifer Sun, Cuthbert Stuckey, Jeanne Shaik, Sason Waskell, Lucy |
author_facet | Yang, Yuting Zhang, Haoming Usharani, Dandamudi Bu, Weishu Im, Sangchoul Tarasev, Michael Rwere, Freeborn Pearl, Naw May Meagher, Jennifer Sun, Cuthbert Stuckey, Jeanne Shaik, Sason Waskell, Lucy |
author_sort | Yang, Yuting |
collection | PubMed |
description | [Image: see text] 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. |
format | Online Article Text |
id | pubmed-4131899 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | American
Chemical Society |
record_format | MEDLINE/PubMed |
spelling | pubmed-41318992015-07-16 Structural and Functional Characterization of a Cytochrome P450 2B4 F429H Mutant with an Axial Thiolate–Histidine Hydrogen Bond Yang, Yuting Zhang, Haoming Usharani, Dandamudi Bu, Weishu Im, Sangchoul Tarasev, Michael Rwere, Freeborn Pearl, Naw May Meagher, Jennifer Sun, Cuthbert Stuckey, Jeanne Shaik, Sason Waskell, Lucy Biochemistry [Image: see text] 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. American Chemical Society 2014-07-16 2014-08-12 /pmc/articles/PMC4131899/ /pubmed/25029089 http://dx.doi.org/10.1021/bi5003794 Text en Copyright © 2014 American Chemical Society Terms of Use (http://pubs.acs.org/page/policy/authorchoice_termsofuse.html) |
spellingShingle | Yang, Yuting Zhang, Haoming Usharani, Dandamudi Bu, Weishu Im, Sangchoul Tarasev, Michael Rwere, Freeborn Pearl, Naw May Meagher, Jennifer Sun, Cuthbert Stuckey, Jeanne Shaik, Sason Waskell, Lucy Structural and Functional Characterization of a Cytochrome P450 2B4 F429H Mutant with an Axial Thiolate–Histidine Hydrogen Bond |
title | Structural and Functional Characterization of a Cytochrome
P450 2B4 F429H Mutant with an Axial Thiolate–Histidine Hydrogen
Bond |
title_full | Structural and Functional Characterization of a Cytochrome
P450 2B4 F429H Mutant with an Axial Thiolate–Histidine Hydrogen
Bond |
title_fullStr | Structural and Functional Characterization of a Cytochrome
P450 2B4 F429H Mutant with an Axial Thiolate–Histidine Hydrogen
Bond |
title_full_unstemmed | Structural and Functional Characterization of a Cytochrome
P450 2B4 F429H Mutant with an Axial Thiolate–Histidine Hydrogen
Bond |
title_short | Structural and Functional Characterization of a Cytochrome
P450 2B4 F429H Mutant with an Axial Thiolate–Histidine Hydrogen
Bond |
title_sort | structural and functional characterization of a cytochrome
p450 2b4 f429h mutant with an axial thiolate–histidine hydrogen
bond |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4131899/ https://www.ncbi.nlm.nih.gov/pubmed/25029089 http://dx.doi.org/10.1021/bi5003794 |
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