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Title: Putidaredoxin Binds to the Same Site on Cytochrome P450cam in the Open and Closed Conformation

Abstract

Cytochrome P450 CYP101A1 (P450cam) hydroxylates camphor by receiving two distinct electrons from its unique reductase, putidaredoxin (Pdx). Upon binding ferric P450cam, Pdx is now known to trigger a conformational change in the enzyme. This Pdx-induced conversion may provide the trigger to coordinate enzyme turnover and protect the enzyme from oxidative damage, so the interactions responsible for this conversion are of significant interest at present. This proposed role for Pdx requires that its interactions with P450cam be different for the open and closed conformations. Here, we show that the binding thermodynamics of Pdx does indeed differ in the predicted way when the conformation of P450cam is held in different states. However, double electron–electron resonance measurements of intermolecular distances in the Pdx/P450cam complex show that the geometry of the complex is nearly identical for the open and closed states of P450cam. These studies show that Pdx appears to make a single distinct interaction with its binding site on the enzyme and triggers the conformational change through very subtle structural interactions.

Authors:
ORCiD logo [1];  [2];  [3]; ORCiD logo [3]; ORCiD logo [3]
  1. Univ. of California, Davis, CA (United States); Max-Planck-Inst. for Biophysical Chemistry, Göttingen (Germany)
  2. Univ. of Oxford (United Kingdom)
  3. Univ. of California, Davis, CA (United States)
Publication Date:
Research Org.:
Univ. of California, Davis, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC); National Institutes of Health (NIH); Engineering and Physical Sciences Research Council (EPSRC)
OSTI Identifier:
1534424
Grant/Contract Number:  
SC0002395; GM41049; FG02-09ER16117; EP/L011972/1
Resource Type:
Accepted Manuscript
Journal Name:
Biochemistry
Additional Journal Information:
Journal Volume: 56; Journal Issue: 33; Journal ID: ISSN 0006-2960
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 59 BASIC BIOLOGICAL SCIENCES; Biochemistry & molecular biology; Interfaces; Peptides and proteins; Ligands; Crystal structure; Conformation

Citation Formats

Liou, Shu-Hao, Myers, William K., Oswald, Jason D., Britt, R. David, and Goodin, David B. Putidaredoxin Binds to the Same Site on Cytochrome P450cam in the Open and Closed Conformation. United States: N. p., 2017. Web. doi:10.1021/acs.biochem.7b00564.
Liou, Shu-Hao, Myers, William K., Oswald, Jason D., Britt, R. David, & Goodin, David B. Putidaredoxin Binds to the Same Site on Cytochrome P450cam in the Open and Closed Conformation. United States. https://doi.org/10.1021/acs.biochem.7b00564
Liou, Shu-Hao, Myers, William K., Oswald, Jason D., Britt, R. David, and Goodin, David B. Tue . "Putidaredoxin Binds to the Same Site on Cytochrome P450cam in the Open and Closed Conformation". United States. https://doi.org/10.1021/acs.biochem.7b00564. https://www.osti.gov/servlets/purl/1534424.
@article{osti_1534424,
title = {Putidaredoxin Binds to the Same Site on Cytochrome P450cam in the Open and Closed Conformation},
author = {Liou, Shu-Hao and Myers, William K. and Oswald, Jason D. and Britt, R. David and Goodin, David B.},
abstractNote = {Cytochrome P450 CYP101A1 (P450cam) hydroxylates camphor by receiving two distinct electrons from its unique reductase, putidaredoxin (Pdx). Upon binding ferric P450cam, Pdx is now known to trigger a conformational change in the enzyme. This Pdx-induced conversion may provide the trigger to coordinate enzyme turnover and protect the enzyme from oxidative damage, so the interactions responsible for this conversion are of significant interest at present. This proposed role for Pdx requires that its interactions with P450cam be different for the open and closed conformations. Here, we show that the binding thermodynamics of Pdx does indeed differ in the predicted way when the conformation of P450cam is held in different states. However, double electron–electron resonance measurements of intermolecular distances in the Pdx/P450cam complex show that the geometry of the complex is nearly identical for the open and closed states of P450cam. These studies show that Pdx appears to make a single distinct interaction with its binding site on the enzyme and triggers the conformational change through very subtle structural interactions.},
doi = {10.1021/acs.biochem.7b00564},
journal = {Biochemistry},
number = 33,
volume = 56,
place = {United States},
year = {2017},
month = {7}
}

Journal Article:
Free Publicly Available Full Text
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Citation Metrics:
Cited by: 18 works
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Figures / Tables:

Figure 1 Figure 1: ITC data for the interaction between Pdx and P450cam in various conformational states. Shown are titrations Pdx into (A) P450cam/metyrapone, which is constrained in the closed conformation, (B) P450cam/camphor, which converts from the closed to open conformation upon binding Pdx, (C) P450cam (substrate-free), which is in the openmore » conformation even in the absence of Pdx, and (D) P450cam/Ada-EG-Dans, which has been shown to populate a conformation of P450cam that is intermediate between open and closed.« less

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