Thermodynamic effects of the alteration of the axial ligand on the unfolding of thermostable cytochrome C.

The role the axial methionine plays in the conformational properties and thermostability of the heme active site has been investigated with the help of site-specific mutations at the axial Met69 position with His (M69H) and Ala (M69A) in thermostable cytochrome c(552) from Thermus thermophilus. Detailed circular dichroism, direct electrochemistry, and other spectroscopic studies have been employed to investigate the thermally induced and GdnHCl-induced unfolding properties of the heme active site of the wild type and the mutants of cytochrome c(552). We observed an unusually high thermodynamic and thermal stability of the M69A mutant compared to that of wild-type cytochrome c(552). However, the M69H mutant exhibited a slightly lower unfolding free energy compared to that of the wild-type protein. The high conformational stability of the M69A mutant was attributed to the presence of residual structure in the unfolded state as well as to the altered conformation in the folded state of this mutant of cytochrome c(552). This study thus supports the view that apart from the folded state, the unfolded state of a protein may also make a significant contribution to the stability of a protein.

Roles of two surface residues near the access channel in the substrate recognition by cytochrome P450cam.

Detailed stopped-flow kinetics of binding of 1R-camphor to cytochrome P450cam has been studied at different temperatures for the wild type as well as for two site specific mutants T192E and S190D of the enzyme, where the surface exposed Threonine and Serine residues were mutated by acidic amino acids. The near-UV and visible circular dichroism spectra as well as the intrinsic fluorescence spectra of the WT and mutant enzymes showed that the mutation of the enzyme did not affect the tertiary structure of the enzyme over the temperature range 4-30 degrees C. The S190D mutation did not show any significant change in the rate constants of the substrate association while they were much lower in the T192E mutant compared to the WT enzyme. The activation energies for substrate association and dissociation processes were determined from the analysis of temperature dependence of the rate constants by the Arrhenius equation over the temperature range 4-19 degrees C. The activation energy for the substrate association was found to be significantly higher in the T192E mutant compared to the S190D mutant or the WT enzyme. The results showed that the Threonine 192 that resides on the F-G loop and directed towards the putative substrate access channel of the enzyme, plays an important role in recognition of the substrate at the surface of the enzyme. These results showed that though the active site of the enzyme resides deep inside the protein matrix, the substrate is recognized at the surface of the enzyme and directed towards the active site through the access channel.

Thermodynamic basis of the thermostability of CYP175A1 from Thermus thermophilus.

Detailed circular dichroism studies have been carried out to monitor thermal as well as denaturant induced unfolding of CYP175A1 from Thermus thermophilus and its mesophilic homologue, CYP101 from Pseudomonas putida. The unfolding midpoint temperatures for tertiary and secondary structures of the substrate-free CYP175A1 were found to be 83.7 degrees C and 87 degrees C respectively, while the corresponding midpoint temperatures for substrate-free CYP101 were at 47.7 degrees C and 51 degrees C respectively. The apparent C(m) value for GdnHCl induced unfolding of secondary structure of CYP175A1 was found to be 2.6M. The thermodynamic stability curves determined from GdnHCl induced unfolding of the enzymes at different temperatures, showed that CYP175A1 had higher free energy compared to CYP101 at temperature >10 degrees C. The results clearly established that the high thermostability of CYP175A1 arises predominantly due to higher enthalpy of the thermostable enzyme compared to CYP101. The larger number of salt bridges was proposed to be responsible for higher enthalpy of stabilization of CYP175A1.

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

The site specific mutants of the thermophilic P450 (P450 175A1 or CYP175A1) were designed to introduce residues that could act as acid-base catalysts near the active site to enhance the peroxidases activity. The Leu80 in the distal heme pocket of CYP175A1 was located at a position almost equivalent to the Glu183 that is involved in stabilization of the ferryl heme intermediate in chloroperoxidase (CPO). The Leu80 residue of CYP175A1 was mutated with histidine (L80H) and glutamine (L80Q) that could potentially form hydrogen bond with hydrogen peroxide and facilitate formation and stabilization of the putative redox intermediate of the peroxidase cycle. The mutants L80H and L80Q of CYP175A1 showed higher peroxidase activity compared to that of the wild type (WT) CYP175A1 enzyme at 25°C. The activity constants (k(cat)) for the L80H and L80Q mutants of CYP175A1 were higher than those of myoglobin and wild type cytochrome b562 at 25° C. The optimum temperature for the peroxidase activity of the WT and mutants of CYP175A1 was ~ 70° C. The rate of catalysis at temperatures above ~ 70° C was higher for L80Q mutant of CYP175A1 compared to that of the well known natural peroxidase, horseradish peroxidase (HRP) that denatures at such high temperature. The peroxidase activities of the mutants of CYP175A1 were maximum at pH 9, unlike that of HRP which is at pH ~5. The results have been discussed in the light of understanding the structure-function relationship of the peroxidase properties of these thermostable heme proteins.