CYP51 is a cytochrome P450 responsible for the oxidative 14α-demethylation of a variety of sterol substrates. This process occurs through a three step reaction, the first two of which are classical hydroxylations which convert the 14α-methyl group first to carboxylalcohol and then to carboxylaldehyde. The third catalytic step releases the methyl group as formic acid and forms a Δ14,15 double bond within the sterol core. The mechanism of this third step has been debated for decades, with proposals involving either an electrophilic “Compound I” or a nucleophilic ferric peroxo anion species. Although human CYP51 and its isoform from <i>Mycobacterium tuberculosis</i> (Mtb) catalyze identical reactions, their crystal structures revealed several species-specific amino acid residues, which are expected to modify the active site environment and as such influence the nature or reactive intermediates states encountered in their enzymatic cycles. In this work, human and Mtb CYP51s were expressed and purified, followed by resonance Raman spectroscopy (rR) to interrogate their active sites and identify structural differences between the two proteins. rR is an especially powerful probe of heme protein structures for both the terminal and equilibrated states in aqueous solutions. It is anticipated that this data will inform the rational design of species selective substrate-based drugs.

Resonance Raman studies of CYP51 isoforms

ACS Spring 2022

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