The catalytic role of the copper ligand H172 of peptidylglycine α-hydroxylating monooxygenase (PHM): A spectroscopic study of the H172A mutant

The spectroscopic characterization of the H172A mutant of peptidylglycine α-hydroxylating monooxygenase (PHM) was undertaken to determine the importance of this Cu_H ligand in the catalytic mechanism of PHM. Mutation of this histidine reduced the activity of the enzyme over 300-fold with little effect on the structure of the oxidized form. However, the reduced enzyme showed a decrease in the average Cu-N(His) distances from 1.96 Å in wild-type PHM to 1.89 Å in H172A associated with a change in the structure of Cu_H from distorted T-shaped planar in the wild type to 2-coordinate in the mutant. Binding of CO was retained at the Cu_M site (similar to wild type), and peptide substrate binding continued to activate a second site for CO binding. Confirmation of this substrate-induced CO binding site at Cu_H was obtained through the observation that loss of the H172 Cu_H ligand caused a 3 cm^-1 blue shift in the v(CO) for this copper carbonyl. Possible mechanistic roles for the H172 ligand are discussed.