Reversible Pressure Deformation of A Thermophilic Cytochrome P450 Enzyme (CYP119) and Its Active-Site Mutants

Abstract

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.

X-ray absorption spectroscopic characterization of a cytochrome P450 compound II derivative

“…CYP119 is a P450 enzyme from the thermophile Sulfolobus solfataricus (7,(21)(22)(23). The enzyme used in this work was expressed in Escherichia coli and was of high purity as judged from the ratio of the absorbance at ϭ 415 nm ( max for Soret band of the heme group) to that at 280 nm, the R/Z ratio.…”

Modification of the heme active site to increase the peroxidase activity of thermophilic cytochrome P450: A rational approach

“…This may increase the electron density at the axial site causing red shift in the Soret band of the mutant enzyme. It has earlier been shown that on application of high pressure to CYP119 leads to a red shift of the Soret band to 425 nm with concomitant changes in the visible bands that are analogous to those observed in the L80H mutant compared with the wild type CYP175A1 [44]. The pressure induced change in the spectrum of CYP119 was proposed to arise due to formation of a 'reversible P420 form' of the enzyme.…”

Kinetic Characterization of Compound I Formation in the Thermostable Cytochrome P450 CYP119

“…The techniques employed were not fast enough to capture the Compound I intermediate on the path to the observed ferryl-radical species. Again, the high concentration of m-CPBA used in these studies leads to degradation of protein, which further complicates data analysis.CYP119 is a thermostable P450 isolated from Sulfolobus solfataricus with a melting temperature of 91°C and remarkable stability to pressure denaturation (25,26). Thermostable * This work was supported by National Institutes of Health Grants GM 31756 and GM 33775.…”

“…CYP119 is a thermostable P450 isolated from Sulfolobus solfataricus with a melting temperature of 91°C and remarkable stability to pressure denaturation (25,26). Thermostable * This work was supported by National Institutes of Health Grants GM 31756 and GM 33775.…”

“…CYP119 is a thermostable P450 isolated from Sulfolobus solfataricus with a melting temperature of 91°C and remarkable stability to pressure denaturation (25,26). Thermostable enzymes are thought to have more rigid active site structures at room temperature than their mesophilic counterparts, which could lead to a slowing of individual steps in the catalytic cycle (27).…”