n this work, we aim to elucidate structural responses to substrate binding as well as pH and P450/P420 equilibrium changes in CYP121. Cytochrome P450 121 (CYP121) is essential for the vitality of Mycobacterium tuberculosis (Mtb), as demonstrated in knockout experiments, and is, therefore, considered a prime target for anti-tubercular therapeutics.1 CYP121 catalyzes a carbon-carbon coupling reaction between two Tyr aromatic rings of its dipeptide cyclo-L-Tyr-L-Tyr (cYY) substrate to form a mycocyclosin product. An interesting aspect of the CYP121 function is that it can undergo a reversible, pH-dependent conversion to its inactive P420 form.2 Since such conversion is typically irreversible in the majority of cytochrome P450, the CYP121 is an ideal model for investigating the structural changes accompanying P420 formation.
The resonance Raman (rR) and UV-Vis spectroscopies are used to investigate the active site structural response to substrate binding, pH changes, and mutation of the catalytically critical R386 residue in the ferric resting state and ferrous-CO ligated forms. Additionally, the effects of these perturbations were evaluated on the enzymatically inactive P420 form.
Substrate binding affects the orientations of the heme peripheral groups as well as the electrostatic environment of the heme active site. The distal R386 reside is critical for substrate binding and its mutation abolishes enzymatic activity and the pH-dependent P450/P420 conversion. The pH changes alter the P450/P420 equilibrium only in ferrous CO forms.
The R386 residue is critical for substrate binding. The mutation of this residue results in the inactivation of the enzymatic activity of CYP121. The R386 mutant does not exhibit reversible P450/P420 conversion, implying the involvement of both proximal and distal active site residues in P450 to P420 conversion.