Aromatase (CYP19) is the target of several therapeutics used for breast cancer treatment and catalyzes the three-step conversion of androgens to estrogens, with an Unusual C-C cleavage reaction in the third step. To better understand the CYP19 reaction, the oxy-ferrous complex of CYP19 with androstenedione Substrate was cryotrapped, characterized by UV-vis spectroscopy, and cryoreduced to generate the next reaction cycle intermediate. EPR analysis revealed that the initial intermediate observed following cryoreduction is the unprotonated g(1) = 2.254 peroxo-ferric intermediate, which is stable Lip to 180 K. Upon gradual cryoannealing, the low-spin (g(1) = 2.39) product complex is formed, with no evidence for accumulation of the g(1) = 2.30 hydroperoxo-ferric intermediate. The relative stabilization of the peroxo-ferric heme and the lack of observed hydroperoxo-ferric heme distinguish CYP19 from other P450s, Suggesting that the Proton delivery pathway is more hindered in CYP19 than in most other P450S. (C) 2009 Elsevier Inc. All rights reserved.