The structure and coordination chemistry of the copper centers in the bifunctional peptidylglycine α-amidating enzyme (α-AE) have been investigated by EPR, EXAFS, and FTIR spectroscopy of a carbonyl derivative. The enzyme contains 2 coppers per 75 kDa protein molecule. Double integration of the EPR spectrum of the oxidized enzyme indicates that 98 ± 13% of the copper is EPR detectable, indicating that the copper centers are located in mononuclear coordination environments. The Cu(II) coordination of the oxidized enzyme is typical of type 2 copper proteins. EXAFS data are best interpreted by an average coordination of 2-3 histidines and 1-20/N (probably O from solvent, Asp or Glu) as equatorial ligands. Reduction causes a major structural change. The Cu(I) centers are shown to be structurally inequivalent since only one of them binds CO. EXAFS analysis of the reduced enzyme data indicates that the non-histidine O/N shell is displaced, and the Cu(I) coordination involves a maximum of 2.5 His ligands together with 0.5 S/CI ligand per copper. The value of ν(CO) (2093 cm-1) derived from FTIR spectroscopy suggests coordination of a weak donor such as methionine, which is supported by a previous observation that the ΔPro-PHM382s mutant M314] is totally inactive. Binding of the peptide substrate N-Ac-Tyr-Val-Gly causes minimum structural perturbation at the Cu(I) centers but appears to induce a more rigid conformation in the vicinity of the S-Met ligand. The unusually intense 8983 eV Cu K-absorption edge feature in reduced and substrate-bound- reduced enzymes is suggestive of a trigonal or digonal coordination environment for Cu(I). A structural model is proposed for the copper centers involving 3 histidines as ligands to Cu1(A) and 2 histidines and 1 methionine as ligands to Cu1(B). However, in view of the intense 8934 eV edge feature and the lack of CO-binding ability, a 2-coordinate structure for Cu(A) is also entirely consistent with the data.
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