Coordination of Hydrogen Peroxide by a Copper Center in Peptidylglycine Alpha-Hydroxylating Monooxygenase (PHM). Structural and Computational Study

Abstract

Many bioactive peptides, such as hormones and growth factors require amidation of the C-terminus for their full biological activity. The enzyme peptidylglycine α-hydroxylating monooxygenase (PHM) carries out the first step of the amidation process, the hydroxylation of peptidylglycine substrates at the Cα position of the terminal glycine. Two non-equivalent copper sites in PHM (CuH and CuM) play distinct roles in the reaction cycle: CuM serves as an oxygen activation and hydrogen abstraction site, while CuH is involved in electron transfer. The proposed mechanism suggests that dioxygen is activated through a two-electron reduction, where each of the copper centers provides a single electron. However, there is an ambiguity regarding the characteristics of the reduced oxygen species in the PHM reaction and the identity of the reactive intermediate. To further investigate the nature of the key intermediates in the PHM cycle we determined the structure of the oxidized form of PHM complexed with hydrogen peroxide. In this 1.95 A resolution structure, the (hydro)peroxide ligand coordinates solely to CuM in a slightly asymmetric side-on mode. The copper-oxygen distances are 1.9 and 2.1 A and the O-O bond 1.5 A. The interatomic O-O distance is characteristic of peroxide/hydroperoxide species, and is significantly longer than the distance typically observed in the superoxide molecule. In addition to the x-ray diffraction studies, we performed DFT calculations using the first coordination sphere of the CuM active site as a model system. Comparison of the relative energies associated with side-on Cu(II)-O22- species and its resonant, end-on structure Cu(I)-O2•- suggests that these two intermediates are significantly populated within the protein environment; this observation has important mechanistic implications.