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Title: Subzero-temperature stabilization and spectroscopic characterization of homogeneous oxyferrous complexes of the cytochrome P450 BM3 (CYP102) oxygenase domain and holoenzyme

Abstract

We describe herein for the first time the formation and spectroscopic characterization of homogeneous oxyferrous complexes of the cytochrome P450 BM3 (CYP102) holoenzyme and heme domain (BMP) at -55 {sup o}C using a 70/30 (v/v) glycerol/buffer cryosolvent. The choice of buffer is a crucial factor with Tris [tris(hydroxymethyl)aminomethane] buffer being significantly more effective than phosphate. The oxyferrous complexes have been characterized with magnetic circular dichroism spectroscopy and the resulting spectra compared to those of the more well-characterized oxyferrous cytochrome P450-CAM. The formation of a stable substrate-bound oxyferrous CYP BM3 holoenzyme, despite the fact that it has the necessary reducing equivalents for turnover, indicates that electron transfer from the flavin domain to the oxyferrous center is very slow at this temperature. The ability to prepare stable homogeneous oxyferrous derivatives of both BMP and the CYP BM3 holoenzyme will enable these species to be used as starting materials for mechanistic investigation of dioxygen activation.

Authors:
 [1];  [1];  [2];  [3]
  1. Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208 (United States)
  2. Department of Chemistry and Biochemistry, University of North Carolina, Greensboro, NC 27402 (United States)
  3. Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208 (United States) and School of Medicine, University of South Carolina, Columbia, SC 29208 (United States). E-mail: dawson@sc.edu
Publication Date:
OSTI Identifier:
20793203
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemical and Biophysical Research Communications; Journal Volume: 338; Journal Issue: 1; Other Information: DOI: 10.1016/j.bbrc.2005.08.078; PII: S0006-291X(05)01791-2; Copyright (c) 2005 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; BUFFERS; COMPLEXES; COMPUTER-AIDED MANUFACTURING; ELECTRON TRANSFER; GLYCEROL; HEME; IRON; MAGNETIC CIRCULAR DICHROISM; PHOSPHATES; SPECTROSCOPY; STABILIZATION; SUBSTRATES

Citation Formats

Perera, Roshan, Sono, Masanori, Raner, Gregory M., and Dawson, John H. Subzero-temperature stabilization and spectroscopic characterization of homogeneous oxyferrous complexes of the cytochrome P450 BM3 (CYP102) oxygenase domain and holoenzyme. United States: N. p., 2005. Web. doi:10.1016/J.BBRC.2005.0.
Perera, Roshan, Sono, Masanori, Raner, Gregory M., & Dawson, John H. Subzero-temperature stabilization and spectroscopic characterization of homogeneous oxyferrous complexes of the cytochrome P450 BM3 (CYP102) oxygenase domain and holoenzyme. United States. doi:10.1016/J.BBRC.2005.0.
Perera, Roshan, Sono, Masanori, Raner, Gregory M., and Dawson, John H. Fri . "Subzero-temperature stabilization and spectroscopic characterization of homogeneous oxyferrous complexes of the cytochrome P450 BM3 (CYP102) oxygenase domain and holoenzyme". United States. doi:10.1016/J.BBRC.2005.0.
@article{osti_20793203,
title = {Subzero-temperature stabilization and spectroscopic characterization of homogeneous oxyferrous complexes of the cytochrome P450 BM3 (CYP102) oxygenase domain and holoenzyme},
author = {Perera, Roshan and Sono, Masanori and Raner, Gregory M. and Dawson, John H.},
abstractNote = {We describe herein for the first time the formation and spectroscopic characterization of homogeneous oxyferrous complexes of the cytochrome P450 BM3 (CYP102) holoenzyme and heme domain (BMP) at -55 {sup o}C using a 70/30 (v/v) glycerol/buffer cryosolvent. The choice of buffer is a crucial factor with Tris [tris(hydroxymethyl)aminomethane] buffer being significantly more effective than phosphate. The oxyferrous complexes have been characterized with magnetic circular dichroism spectroscopy and the resulting spectra compared to those of the more well-characterized oxyferrous cytochrome P450-CAM. The formation of a stable substrate-bound oxyferrous CYP BM3 holoenzyme, despite the fact that it has the necessary reducing equivalents for turnover, indicates that electron transfer from the flavin domain to the oxyferrous center is very slow at this temperature. The ability to prepare stable homogeneous oxyferrous derivatives of both BMP and the CYP BM3 holoenzyme will enable these species to be used as starting materials for mechanistic investigation of dioxygen activation.},
doi = {10.1016/J.BBRC.2005.0},
journal = {Biochemical and Biophysical Research Communications},
number = 1,
volume = 338,
place = {United States},
year = {Fri Dec 09 00:00:00 EST 2005},
month = {Fri Dec 09 00:00:00 EST 2005}
}