title: Analysis of the structural stability of the multidomain enzyme flavocytochrome P-450 BM3.
abstract: The unfolding and refolding of flavocytochrome P-450 BM3 and its constituent haem and flavin domains have been analysed, using guanidinium chloride (GdnHCl) as a denaturant. Enzyme activities are lost at GdnHCl concentrations too low to cause major changes in secondary structure (0.1-0.5 M). The losses are primarily due to time-dependent FMN removal. Fluorescence and visible CD spectroscopies show that FMN dissociation is complete by 0.7 M GdnHCl, whereas FAD removal is complete by 1.5 M GdnHCl. Limited regain of activity is achieved by dilution of enzyme from solutions of < or = 0.75 M GdnHCl into fresh buffer. Supplementation of GdnHCl-free assay media with flavins (FAD and FMN) causes small additional regains in flavin domain (cytochrome-c reductase) activity lost at low [GdnHCl]. However, flavin addition during the denaturation step affords greater protection against inactivation, suggesting that conformational changes may occur subsequent to flavin loss and that these changes are not readily reversed on dilution of GdnHCl. Loss of catalytically competent haem ligation occurs over the same [GdnHCl] range for P-450 BM3 and its haem domain. In both cases, the 'denatured' P-420 form accumulates in the reduced/carbon monoxide-bound visible spectrum from 0.5 to 2 M GdnHCl. Secondary structure loss also occurs over similar [GdnHCl] ranges for P-450 BM3 and its two domains (80-90% lost from 0.5-3 M GdnHCl), indicating that there is little mutual stabilisation of domains in the holoenzyme. Differential scanning calorimetry measurements support this conclusion, but show that the haem domain is more thermostable than the flavin domain.
authors.affiliation: Division of Biochemistry and Molecular Biology, University of Glasgow, UK. gbca49@udcf.gla.ac.uk
authors.forename: A W
authors.initials: AW
authors.lastname: Munro
authors.forename: J G
authors.initials: JG
authors.lastname: Lindsay
authors.forename: J R
authors.initials: JR
authors.lastname: Coggins
authors.forename: S M
authors.initials: SM
authors.lastname: Kelly
authors.forename: N C
authors.initials: NC
authors.lastname: Price
mesh_terms: D001410:Bacillus megaterium / Q000201:enzymology; D001426:Bacterial Proteins / Q000737:chemistry* / Q000187:drug effects; D001665:Binding Sites; D002942:Circular Dichroism; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000187:drug effects; D005486:Flavin Mononucleotide / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000187:drug effects; D009251:NADPH-Ferrihemoprotein Reductase; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013057:Spectrum Analysis
chemical_list: D001426:Bacterial Proteins; D006146:Guanidines; D011993:Recombinant Fusion Proteins; D005182:Flavin-Adenine Dinucleotide; D005486:Flavin Mononucleotide; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D009251:NADPH-Ferrihemoprotein Reductase; C052423:flavocytochrome P450 BM3 monoxygenases; D019791:Guanidine