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

The human sterol 14 alpha-demethylases (CYP51, CYP is an abbreviation for cytochrome P450) catalyze three-step oxidative removal of 14 alpha-methyl group of lanosterol by first forming an alcohol, then an aldehyde, and finally conducting a C-C 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 hy-droxylase 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, never-theless, 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 sub-strates. Moreover, a positive polar environment around the exogenous diatomic ligand is detected, providing insight into the mechanism of this essential C-C bond cleavage reaction.

Automated summary

This study investigates the active site structure of CYP51 enzyme in the presence of its hydroxylase and lyase substrates using Resonance Raman spectroscopy and Nanodisc technology. The results show that the hydroxyl group of the lyase substrate interacts directly with the iron center, while a water ligand coordinated to the heme iron is retained, leading to a low degree of spin conversion in CYP51. The nanodisc-incorporated assemblies provide more well-defined active site responses and induce a larger spin conversion.