Intramolecular heme ligation of the cytochrome P450 2C9 R108H mutant demonstrates pronounced conformational flexibility of the B-C loop region: Implications for substrate binding

Roberts, Arthur G., Cheesman, Matthew J., Primak, Andrew, Bowman, Michael K., Atkins, William M. and Rettie, Allan E. (2010) Intramolecular heme ligation of the cytochrome P450 2C9 R108H mutant demonstrates pronounced conformational flexibility of the B-C loop region: Implications for substrate binding. Biochemistry, 49 40: 8700-8708. doi:10.1021/bi100911q

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Author Roberts, Arthur G.
Cheesman, Matthew J.
Primak, Andrew
Bowman, Michael K.
Atkins, William M.
Rettie, Allan E.
Title Intramolecular heme ligation of the cytochrome P450 2C9 R108H mutant demonstrates pronounced conformational flexibility of the B-C loop region: Implications for substrate binding
Journal name Biochemistry   Check publisher's open access policy
ISSN 0006-2960
1943-295X
Publication date 2010-10-12
Sub-type Article (original research)
DOI 10.1021/bi100911q
Volume 49
Issue 40
Start page 8700
End page 8708
Total pages 9
Place of publication Washington, DC, United States
Publisher American Chemical Society
Collection year 2011
Language eng
Formatted abstract
A previous study [Dickmann, L., et al. (2004) Mol. Pharmacol. 65, 842-850] revealed some unusual properties of the R108H mutant of cytochrome P450 2C9 (CYP2C9), including elevated thermostability relative to that of CYP2C9, as well as a UV-visible absorbance spectrum that was indicative of nitrogenous ligation to the heme iron. In our study, size-exclusion chromatography and UV-visible absorbance spectroscopy of CYP2C9 R108H monomers demonstrated that nitrogen ligation is indeed intramolecular. Pulsed electron paramagnetic resonance of CYP2C9 R108H monomers showed that a histidine is most likely bound to the heme as previously hypothesized. An energy-minimized model of the R108H mutant maintained a CYP fold, despite substantial movement of several loop regions of the mutant, and, therefore, represents an extreme example of a closed conformation of the enzyme. Molecular dynamics (MD) simulations of CYP2C9 were performed to study the range of energetically accessible CYP2C9 conformations. These in silico studies showed that the B-C loop region of CYP2C9 moves away from the heme to a position resembling the putative open conformation described for rabbit CYP2B4. A model involving the movement of the B-C loop region and R108 between the open and closed conformations of CYP2C9 is presented, which helps to explain the enzyme's ability to regio- and stereospecifically metabolize some ligands while allosterically activating others.
© 2010 American Chemical Society.
Keyword Amino acids
Enzymes
Molecular dynamics
Monomers
Paramagnetic resonance
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2011 Collection
School of Biomedical Sciences Publications
 
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Citation counts: TR Web of Science Citation Count  Cited 9 times in Thomson Reuters Web of Science Article | Citations
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Created: Tue, 22 Mar 2011, 08:55:58 EST by Bacsweet Kaur on behalf of School of Biomedical Sciences