Revealing substrate-induced structural changes in active site of human CYP51 in the presence of its physiological substrates

The human sterol 14α-demethylases (CYP51, CYP is an abbreviation for cytochrome P450) catalyze three-step oxidative removal of 14α-methyl group of lanosterol by first forming an alcohol, then an aldehyde, and finally conducting a CC bond cleavage reaction. This present study utilizes a combination of Resonance Raman spectroscopy and Nanodisc technology to probe the active site structure of CYP51 in the presence of its hydroxylase and lyase substrates. Ligand-binding induced partial low-to-high-spin conversion is observed by applying electronic absorption spectroscopy and Resonance Raman (RR) spectroscopy. This low degree of spin conversion of CYP51 is contributed by the retention of the water ligand coordinated to the heme iron as well as direct interaction between the hydroxyl group of lyase substrate and the iron center. No significant changes in active site structure are found between detergent-stabilized CYP51 and nanodisc-incorporated CYP51, nevertheless, it is demonstrated that nanodisc-incorporated assemblies provide much more well-defined active site RR spectroscopic responses, which induces a larger conversion from low-to-high-spin state in presence of the substrates. Moreover, a positive polar environment around the exogenous diatomic ligand is detected, providing insight into the mechanism of this essential CC bond cleavage reaction. Resonance Raman spectra of Nanodisc incorporated human sterol 14α-demethylase CO adducts. [Display omitted] •Nanodisc-incorporated 14α-demethylase resulted in a larger spin state conversion upon binding of substrates. comparing to detergent-solubilized CYP51.•Binding of the lanosterol induced a partial -spin state transition that attributed to the retention of the heme aqua ligand.•The addition of substrates to CO derivative of 14α-demethylase resulted in a polar environment around the CO ligand.