Depicting the proton relay network in human aromatase: New insights into the role of the alcohol-acid pair

Human aromatase is the cytochrome P450 catalyzing the conversion of androgens into estrogens in a three steps reaction essential to maintain steroid hormones balance. Here we report the capture and spectroscopic characterization of its compound I (Cpd I), the main reactive species in cytochromes P450. The typical spectroscopic transitions indicating the formation of Cpd I are detected within 0.8 s when mixing aromatase with meta-chloroperoxybenzoic acid. The estrogen product is obtained from the same reaction mixture, demonstrating the involvement of Cpd I in aromatization reaction. Site-directed mutagenesis is applied to the acid-alcohol pair D309 and T310 and to R192, predicted to be part of the proton relay network. Mutants D309N and R192Q do not lead to Cpd I with an associated loss of activity, confirming that these residues are involved in proton delivery for Cpd I generation. Cpd I is captured for T310A mutant and shows 2.9- and 4.4-fold faster rates of formation and decay, respectively, compared to wild-type (WT). However, its activity is lower than the WT and a larger amount of H2O2 is produced during catalysis, indicating that T310 has an essential role in proton gating for generation of Cpd 0 and Cpd I and for their stabilization. The data provide new evidences on the role of threonine belonging to the conserved acid-alcohol pair and known to be crucial for oxygen activation in cytochromes P450.

Automated summary

This study reports the capture and spectroscopic characterization of compound I (Cpd I) of human aromatase, highlighting its importance in aromatization reaction. Mutagenesis experiments confirm the involvement of specific residues in proton delivery for Cpd I generation. The data provide new evidences on the role of threonine in oxygen activation in cytochromes P450.