Aims – This work aims to elucidate the structural differences between the cytochromes P450 CYP51 from Human (Hu) and Mycobacterium tuberculosis (Mtb). Cytochromes P450 are enzymes that contain a cysteine-ligated heme prosthetic group, allowing them to generate reactive oxidizing species by binding molecular oxygen. CYP51 is hypothesized to be one of the oldest P450s, and is observed across biological kingdoms. In all cases CYP51 cytochromes perform a 14-α demethylation reaction on a sterol substrate, despite low overall sequence homology (only 33% between Mtb and Hu CYP51).1 These differences in amino acid sequence correspond to key structural differences in the active sites of these proteins, which may be exploited to design organism-specific drugs.
Methods – UV-Vis absorption spectroscopy (Uv-Vis) and resonance Raman spectroscopy (rR) have been used to probe the active sites of both proteins in different oxidation and ligation states (CO, CN-), at acidic and basic pH, and in a presence of a bound substrate analog (estriol).
Results – The spectroscopic studies of the CO and CN- adducts of Mtb and Hu enzymes revealed subtle, but functionally important structural differences between both proteins. The CN- binding experiments showed significantly higher affinity for Mtb CYP51 compared to Hu CYP51. The addition of substrate to Mtb CYP51 resulted in partial spin-state conversion for Mtb CYP51, but not for Hu CYP51. Finally, the conversion between active “P450” and the inactive “P420” forms upon reduction and binding of carbon monoxide (CO) showed pH and species dependences.
Conclusion – These studies revealed subtle but functionally important structural differences between two CYP51 cytochromes. While both enzymes perform the same C-C bond cleavage reaction, the active sides variations might affect their overall enzymatic activity and stability of reactive intermediate states.