The binuclear copper state of peptidylglycine monooxygenase visualized through a selenium-substituted peptidyl-homocysteine complex.

Bioactive peptides generally require post-translational processing to convert them to their fully active forms. Peptidylglycine monooxygenase (PHM) is a copper-dependent enzyme that catalyzes C-alpha hydroxylation of a glycine-extended pro-peptide, a critical post-translational step in peptide amidation. A canonical mechanism based on experimental and theoretical considerations proposes that molecular oxygen reacts at the mononuclear CuM-center to form a reactive Cu(II)-superoxo intermediate capable of H-atom abstraction from the peptidyl substrate, followed by long range ET from the CuH center positioned 11 Å away across a solvent-filled cleft. However, recent data has challenged this mechanism, suggesting instead that an "open-to-closed" conformational transition brings the copper centers closer to facilitate reaction at a binuclear copper site. Here we present direct observations of an enzyme-bound binuclear copper species, which was enabled by the use of an Ala-Ala-Phe-homoselenocysteine (hSeCys) species. EXAFS, UV/vis, and EPR studies are used to show that this reagent reacts with the oxidized enzyme to form a novel mixed valence entity which is subtly different from that observed previously for the S-peptidyl complex (K. W. Rush, K. A. S. Eastman, E. F. Welch, V. Bandarian and N. J. Blackburn,