Reversible pressure deformation of a thermophilic cytochrome P450 enzyme (CYP119) and its active-site mutants.
The pressure stability of the thermophilic CYP119 from Sulfolobus solfataricus and its active-site Thr213 and Thr214 mutants was investigated. At 20 degrees C and pH 6.5, the protein undergoes a reversible P450-to-P420 inactivation with a midpoint at 380 MPa and a reaction volume change of -28 mL/mol. The volume of activation of the process was -9.5 mL/mol. The inactivation transition was retarded, and the absolute reaction volume was decreased by increasing temperature or by mutations that decrease the size of the active-site cavity. High pressure affected the tryptophan fluorescence yield, which decreased by about 37% at 480 MPa. The effect was reversible and suggested considerable contraction of the protein. Aerobic decomposition of iron-aryl complexes of the CYP119 T213A mutant under increasing hydrostatic pressure resulted in variation of the N-arylprotoporphyrin-IX regioisomer (N(B):N(A):N(C):N(D)) adduct pattern from 39:47:07:07 at 0.1 MPa to 23:36:14:27 at 400 MPa. Preincubation of the protein at 400 MPa followed by complex formation and decomposition gave the same regioisomer distribution as untreated protein. The results indicate that the protein is reversibly inactivated by pressure, in contrast to the irreversible inactivation of P450(cam) and other P450 enzymes, and that this inactivation process is modulated by changes in the active-site cavity dimensions.
Tschirret-Guth
R A
RA
Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, CA 94143-0446, USA.
Koo
L S
LS
Hoa
G H
GH
Ortiz De Montellano
P R
PR
D019843:Archaeal Proteins; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli; D005453:Fluorescence; D009154:Mutation; D010105:Oxygenases / Q000737:chemistry* / Q000235:genetics; D011312:Pressure; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D013054:Spectrophotometry, Atomic; D013696:Temperature
D019843:Archaeal Proteins; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; C408874:CYP119 protein, Sulfolobus solfataricus