A thermostable cytochrome P450 from Sulfolobus solfataricus

Cytochromes P450 are responsible for the regio- and stereospecific monoxygenation of a wide range of compounds. This work describes the spectroscopic and X-ray crystallographic studies of a thermostable P450, CYP119, cloned from the acidothermophilic archaeon Sulfolobus solfataricus. CYP119 was found to have the characteristic spectroscopic properties and triangular fold typical for this class of isozymes, even though CYP119 is shorter in amino acid sequence and has a low sequence homology to other known P450 cytochromes. Optical spectroscopy was used to monitor the changes of the local heme environment when perturbed by temperature, pressure, pH, and the presence of fatty acids. CYP119 shows increased resistance to P420 formation. Differential scanning calorimetry has shown CYP119 to be more thermostable than its mesophilic counterparts with a 40°C higher melting temperature.; Crystallographic studies have revealed the placement of conserved key catalytic residues and solvent in the active site that are important in the P450 dioxygen scission reaction. CYP119 is found to display a mobile F-G helix and loop region that undergoes a significant conformational change upon binding of medium and large ligands and may reflect a natural motion independent of ligand binding. The CYP119 porphyrin is non-planar, with a “ruffling” and “saddling” of the porphyrin heterocycle. Additionally, the iron favors an in-plane position, which correlates with the spectroscopic data showing that CYP119 prefers the low-spin conformational state. This favoring of the low-spin state may have an effect on the catalytic properties of CYP119, for example the fast autoxidation rate of the oxyferrous enzyme. The overall protein structure was found to have a unique distribution of charge, which could potentially affect the normal electrostatic interaction with redox partners involved in electron transfer to the P450 cytochromes. The crystal structures of CYP119 indicate that increased stability may be due to a combination of increased factors involved in stabilizing secondary and tertiary structural interactions, such as aromatic stacking, increased salt link networks, short strong hydrogen bonds, and increased secondary structure due to shorter loops. These insights into local and globular protein stability should aid in the optimization of proteins for drug design and bio-organic chemical synthesis.