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Title: Active-Site Hydration and Water Diffusion in Cytochrome P450cam: A Highly Dynamic Process

Abstract

Long-timescale molecular dynamics simulations (300 ns) are performed on both the apo- (i.e., camphor-free) and camphor-bound cytochrome P450cam (CYP101). Water diffusion into and out of the protein active site is observed without biased sampling methods. During the course of the molecular dynamics simulation, an average of 6.4 water molecules is observed in the camphor-binding site of the apo form, compared to zero water molecules in the binding site of the substrate-bound form, in agreement with the number of water molecules observed in crystal structures of the same species. However, as many as 12 water molecules can be present at a given time in the camphor-binding region of the active site in the case of apo-P450cam, revealing a highly dynamic process for hydration of the protein active site, with water molecules exchanging rapidly with the bulk solvent. Water molecules are also found to exchange locations frequently inside the active site, preferentially clustering in regions surrounding the water molecules observed in the crystal structure. Potential-of-mean-force calculations identify thermodynamically favored trans-protein pathways for the diffusion of water molecules between the protein active site and the bulk solvent. Binding of camphor in the active site modifies the free-energy landscape of P450cam channels toward favoringmore » the diffusion of water molecules out of the protein active site.« less

Authors:
 [1];  [1]
  1. ORNL
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Laboratory Directed Research and Development (LDRD) Program
OSTI Identifier:
1037664
DOE Contract Number:
DE-AC05-00OR22725
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biophysical Journal; Journal Volume: 101; Journal Issue: 6
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; CAMPHOR; CRYSTAL STRUCTURE; CYTOCHROMES; DIFFUSION; FREE ENERGY; HYDRATION; PROTEINS; SAMPLING; SIMULATION; WATER

Citation Formats

Miao, Yinglong, and Baudry, Jerome Y. Active-Site Hydration and Water Diffusion in Cytochrome P450cam: A Highly Dynamic Process. United States: N. p., 2011. Web. doi:10.1016/j.bpj.2011.08.020.
Miao, Yinglong, & Baudry, Jerome Y. Active-Site Hydration and Water Diffusion in Cytochrome P450cam: A Highly Dynamic Process. United States. doi:10.1016/j.bpj.2011.08.020.
Miao, Yinglong, and Baudry, Jerome Y. 2011. "Active-Site Hydration and Water Diffusion in Cytochrome P450cam: A Highly Dynamic Process". United States. doi:10.1016/j.bpj.2011.08.020.
@article{osti_1037664,
title = {Active-Site Hydration and Water Diffusion in Cytochrome P450cam: A Highly Dynamic Process},
author = {Miao, Yinglong and Baudry, Jerome Y},
abstractNote = {Long-timescale molecular dynamics simulations (300 ns) are performed on both the apo- (i.e., camphor-free) and camphor-bound cytochrome P450cam (CYP101). Water diffusion into and out of the protein active site is observed without biased sampling methods. During the course of the molecular dynamics simulation, an average of 6.4 water molecules is observed in the camphor-binding site of the apo form, compared to zero water molecules in the binding site of the substrate-bound form, in agreement with the number of water molecules observed in crystal structures of the same species. However, as many as 12 water molecules can be present at a given time in the camphor-binding region of the active site in the case of apo-P450cam, revealing a highly dynamic process for hydration of the protein active site, with water molecules exchanging rapidly with the bulk solvent. Water molecules are also found to exchange locations frequently inside the active site, preferentially clustering in regions surrounding the water molecules observed in the crystal structure. Potential-of-mean-force calculations identify thermodynamically favored trans-protein pathways for the diffusion of water molecules between the protein active site and the bulk solvent. Binding of camphor in the active site modifies the free-energy landscape of P450cam channels toward favoring the diffusion of water molecules out of the protein active site.},
doi = {10.1016/j.bpj.2011.08.020},
journal = {Biophysical Journal},
number = 6,
volume = 101,
place = {United States},
year = 2011,
month = 1
}
  • Orientation selective and multiple frequency ESEEM experiments on substrate free cyctochrome P450cam (CP450cam) with {sup 17}O-enriched water are reported. The {sup 17}O ESEEM frequencies were obtained from Fourier transformation of the ratio of ESEEM waveforms of CP450cam with enriched water and CP450cam with non-enriched water. Numerical simulations were carried out to determine the isotropic and the anisotropic hyperfine interactions and the quadrupole interaction of the {sup 17}O. From the magnitude (e{sup 2}qQ/h = 6.6 MHz) and asymmetry ({eta} = 0.95) of the {sup 17}O quadrupole interaction, we conclude that the distal axial ligand of Fe{sup 3+} in the CP450 hememore » is a water molecule. Moreover, from the orientation of the {sup 17}O quadrupole tensor, the orientation of the water molecule was found to be confined (within {+-}10{degree}) with respect to the Fe-N directions within the heme plane. Two possible sets of isotropic and anisotropic components of the {sup 17}O hyperfine interaction, ({+-}2.6, {+-}10.3 MHz) and ({+-}0.4, {+-}1.8 MHz), were found to satisfactorily reproduce the experimental results. Both sets indicate very small {sup 17}O hyperfine couplings which is consistent with the unpaired electron residing predominately in the d{sub yz} orbital of the Fe{sup 3+}. Ligand field models for each set are presented. 50 refs., 11 figs., 1 tab.« less
  • Water proton relaxation rates of various complexes of cholesterol side chain cleavage cytochrome P-450 (P-450/sub scc/) were investigated to gain information about the structure and dynamics of the steroid binding site. In all cases bulk water protons were found to be in rapid exchange with protons near the paramagnetic Fe/sup 3 +/ center, and the long electron spin relaxation time of the heme iron, tau/sub s/ approx. 0.3 ns, resulted in fast relaxation rates. For the steroid-free enzyme, the closest approach of exchangeable protons is approx. 2.5 A, a distance consistent with a water molecule binding directly to the hememore » iron or rapidly exchanging with a coordinated ligand. When cholesterol was bound, the distance increased to approx. 4 A, indicative of displacement of water from the immediate coordination sphere of the heme but still in close proximity to the active site. For the complex with (22R)-22-hydroxycholesterol, a distance of approx. 2.7 A is observed, suggesting a reorganization of the active site when this intermediate is formed from cholesterol. Complexes of P-450/sub scc/ with the competitive inhibitors (22R)-22-aminocholesterol, 22-amino-23,24-bisnor-5-cholen-3..beta..-ol, or (20R)-20-phenyl-5-pregnene-3..beta..,20-diol, also yielded distances of approx. 2.5 A and reveal no effect of side chain size on access of protons to the heme. In the nitrogen-coordinated amino-steroid complexes, the distances observed indicate solvent proton exchange with the heme-bound nitrogen ligand. In contrast to cytochrome P-450/sub cam/, in which water is excluded from the heme center in the substrate complex, protons have rapid access to regions near the active site of several steroid complexes of P-450/sub scc/. This suggests that the active site of P-450/sub scc/ may be open to solvent and that solvent water molecules, rather than acid/base groups in the active site, may provide the protons required during the monooxygenation reaction cycle.« less
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