title: Structural investigations on the coordination environment of the active-site copper centers of recombinant bifunctional peptidylglycine alpha-amidating enzyme.
abstract: 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 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 1-2 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/CI ligand per copper. The value of v(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 delta Pro-PHM382s mutant M314I 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 CuIA and 2 histidines and 1 methionine as ligands to CuIB. However, in view of the intense 8934 eV edge feature and the lack of CO-binding ability, a 2-coordinate structure for CuA is also entirely consistent with the data.
authors.affiliation: Department of Chemistry, Biochemistry, and Molecular Biology, Oregon Graduate Institute of Science & Technology, Portland 97291-1000, USA.
authors.forename: J S
authors.initials: JS
authors.lastname: Boswell
authors.forename: B J
authors.initials: BJ
authors.lastname: Reedy
authors.forename: R
authors.initials: R
authors.lastname: Kulathila
authors.forename: D
authors.initials: D
authors.lastname: Merkler
authors.forename: N J
authors.initials: NJ
authors.lastname: Blackburn
mesh_terms: D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002248:Carbon Monoxide / Q000378:metabolism; D003001:Cloning, Molecular; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D006224:Cricetinae; D004578:Electron Spin Resonance Spectroscopy; D006639:Histidine / Q000737:chemistry / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000378:metabolism; D009097:Multienzyme Complexes; D009842:Oligopeptides / Q000378:metabolism; D010084:Oxidation-Reduction; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared; D013057:Spectrum Analysis; D014965:X-Rays
chemical_list: D009097:Multienzyme Complexes; D009842:Oligopeptides; D011994:Recombinant Proteins; D006639:Histidine; D003300:Copper; D002248:Carbon Monoxide; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase