Subzero-temperature stabilization and spectroscopic characterization of homogeneous oxyferrous complexes of the cytochrome P450 BM3 (CYP102) oxygenase domain and holoenzyme.
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 degrees 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.
Perera
Roshan
R
Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208, USA.
Sono
Masanori
M
Raner
Gregory M
GM
Dawson
John H
JH
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D020134:Catalytic Domain; D002942:Circular Dichroism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism; D004795:Enzyme Stability; D005615:Freezing; D007501:Iron / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism; D009251:NADPH-Ferrihemoprotein Reductase; D010100:Oxygen / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry; D013816:Thermodynamics
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D007501:Iron; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; D010100:Oxygen