Substitution of iron with cobalt in the prosthetic group of bacterial cytochrome P450: Effects on the stability and structure of the protein

The origin of thermostability of the heme monooxygenase from Therms thermophilus, CYP175A1 has been investigated using multi-wavelength circular dichroism (CD) spectroscopy at different temperatures. The far-UV and visible CD studies were carried out on WTCYP175A1, reduced WTCYP175A1 (i.e. Fe(II)-WTCYP175A1), apoCYP175A1, and the cobalt protoporphyrin (CoP) bound apoprotein (CoPCYP175A1) to determine the thermostability of the secondary and tertiary structure of the variants of the enzyme. Non-linear least squares analyses of the multi-wavelength CD data were used to determine the midpoint unfolding temperature (T-m), and the associated thermodynamic parameters for unfolding of the enzyme. The midpoint temperature (T-m) for thermal unfolding of WTCYP175A1 was much higher than that of the reduced enzyme (Fe(II)-WTCYP175A1), which was higher than that of the CoP bound analogue, while the T-m for the apoprotein was even lower than the CoPCYP175A1. The temperature for maximum stability (T-s) also followed the order WTCYP175A1 > Fe(II)-WTCYP175A1 approximate to CoPCYP175A1 > apoCYP175A1 supporting that the binding of the metal prosthetic group indeed have important role on the overall stability of the protein. However, the maximum free energy of unfolding (Delta G(s)) of the secondary structure of the protein remained almost the same for all variants of CYP175A1. These studies thus show that weak non-bonding interactions such as hydrogen bonding and metal coordination play critical role in imparting high thermostability of the overall structure of the thermostable enzyme, CYP175A1. The thermostability of tertiary structure of the active site of the enzyme was also shown to depend on the nature of binding of the metal cofactor to the protein matrix.