Structural Investigations on the Coordination Environment of the Active-Site Copper Centers of Recombinant Bifunctional Peptidylglycine α-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.

O 2 activation by binuclear Cu sites: Noncoupled versus exchange coupled reaction mechanisms

“…Because J is small for the noncoupled Cu M and Cu H sites, the (H DA ) 2 between the two Cu centers also must be small. Significant geometry changes between the reduced and oxidized forms of the Cu M and Cu H sites have been observed experimentally (10)(11)(12)29) and are also found in calculated structures (13,16), which suggest a large reorganization energy ( inner ) is also associated with their redox reactions (Fig. 2, A, B, C, and D, respectively).…”

“…2, A, B, C, and D, respectively). [Note that the crystal structures of PHM (6,8) did not resolve significant differences between the oxidized and the reduced proteins, whereas EXAFS results showed significant geometry changes upon redox (10)(11)(12). EXAFS studies are more accurate in determining the metal-ligand bond lengths and differentiating Cu oxidation states.]…”

“…Structural information from crystallographic (6,8) and EXAFS (extended x-ray absorption fine structure) studies (10)(11)(12), combined with recent advances in spectroscopic This paper was submitted directly (Track II) to the PNAS office. Fig.…”

Cell Type-specific Storage of Dopamine β-Monooxygenase

“…B, for rat DBM the signal sequence (SS), the disulfide bridges, and N-glycosylation sites (dark circles) are indicated (15,16). The region of homology between DBM and PHM as well as the specificity of the DBM antibody (Ab2047) are also indicated.…”

“…1B) (6,14). Interestingly, this region contains four conserved disulfide bridges and six conserved copper ligands (15,16). Despite these similarities, the topologies of DBM and PAM are very different (Fig.…”