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

UNCG Author/Contributor (non-UNCG co-authors, if there are any, appear on document)
Gregory M. Raner, Associate Professor and Graduate Director (Creator)
Institution
The University of North Carolina at Greensboro (UNCG )
Web Site: http://library.uncg.edu/

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 °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.

Additional Information

Publication
Biochemical and Biophysical Research Communications 202, 365-371 (2005).
Language: English
Date: 2005
Keywords
Mono-oxygenase, Dioxygen activation, Oxyferrous heme iron, Magnetic circular dichroism, Cryoenzymology, Cysteinate-ligated heme iron, Cytochrome P450 BM3