Structural investigations on the coordination environment of the active-site copper centers of recombinant bifunctional peptidylglycine {alpha}-amidating enzyme
- Oregon Graduate Institute of Science & Technology, Portland, OR (United States); and others
The structure and coordination chemistry of the copper centers in the bifunctional peptidylglycine {alpha}-amidating enzyme ({alpha}-AE) have been investigated by EPR, EXAFS, and FTIR spectroscopy of a carbonyl derivative. The enzyme contains 2 copper per 75 kDa protein molecule. Double integration of the EPR spectrum of the oxidized enzyme indicates that 98 {plus_minus} 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 1-2 O/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 nonhistidine O/N shell is displaced, and the Cu(I) coordination involves a maximum of 2.5 His ligands together with 0.5 S/Cl ligand per copper. The value of {nu}(CO) (2093 cm{sup {minus}1}) derived from FTIR spectroscopy suggests coordination of a weak donor such as methionine, which is supported by a previous observation that the {Delta}Pro-PHM382s mutant M{sup 314}I 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 digonal coordination environment for Cu(I). A structural model is proposed for the copper centers involving 3 histidines as ligands to Cu{sub A}{sup 1} and 2 histidines and 1 methionine as ligands to Cu{sub B}{sup 1}. 57 refs., 7 figs., 3 tabs.
- OSTI ID:
- 501779
- Journal Information:
- Biochemistry (Eaton), Journal Name: Biochemistry (Eaton) Journal Issue: 38 Vol. 35; ISSN 0006-2960; ISSN BICHAW
- Country of Publication:
- United States
- Language:
- English
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