Peptide and peptide-protein models of the plastocyanin copper-binding site.

Type 1 "blue" copper proteins such as plastocyanin are of interest due to their unusual active site geometries, elevated redox potentials, and rapid electron-transfer rates. Previously, the Cu-coordination properties of small peptide models of the Poplar plastocyanin active site metal binding loop, containing His/Cys/Met putative ligands ('BCPA' peptides), were found to exhibit slight shifts towards the tetragonal distortion supported in the protein active site. Here, several additional Cu-peptide models are explored with spectroscopic and electrochemical measurements and computational methods. Changes to the putative plastocyanin Met ligand in BCPA peptides and Cu coordination properties of peptides fused to maltose binding protein (MBP) are investigated. While Cu(II/I) midpoint reduction potentials of the BCPA class were all in the +130-150 mV (vs. NHE) range, X-band EPR studies of the Met-substituted models indicate that Met does not coordinate in Cu(II)-BCPA. It is proposed that either a hydroxide or a deprotonated amide group binds to the open equatorial site. To examine portability of a Cu-peptide motif, an expanded model containing both the plastocyanin Cu-binding loop and the adjacent β-hairpin stem (peptide PLC) was expressed as a fusion to MBP. UV-vis spectroscopy showed a species in low concentration with a thiolate-Cu(II) LMCT band red-shifted ∼30 nm from that in Cu(II)-BCPA. Cu coordinated to MBP fusions of other BCPA peptides exhibited reduction potentials and EPR spectra that differed significantly from the isolated Cu-peptides, suggesting interactions with MBP. The suite of Cu-peptide models studied here exhibit estimated reduction potentials ranging from ∼140 to ∼80 mV vs. NHE.