Design and spectroscopic characterization of peptide models for the plastocyanin copper-binding loop.
The Cu(II)- and Co(II)-binding properties of two peptides, designed on the basis of the active site sequence and structure of the blue copper protein plastocyanin, are explored. Peptide BCP-A, Ac-Trp-(Gly)(3)-Ser-Tyr-Cys-Ser-Pro-His-Gln-Gly-Ala-Gly-Met-(Gly )(3)-His-(Gly)(2)-Lys-CONH(2), conserves the Cu-binding loop of plastocyanin containing three of the four copper ligands and has a flexible (Gly)(3) linker to the second His ligand. Peptide BCP-B, Ac-Trp-(Gly)(3)-Cys-Gly-His-Gly-Val-Pro-Ser-His-Gly-Met-Gly-CONH(2), contains all four blue copper ligands, with two on either side of a beta-turn. Both peptides form 1:1 complexes with Cu(II) through His and Cys ligands. BCP-A, the ligand loop, binds to Cu(II) in a tetrahedrally distorted square plane with axial solvent ligation, while BCP-B-Cu(II) has no tetrahedral distortion in aqueous solution. In methanolic solution, distortion of the square plane is evident for both BCP-Cu(II) complexes. Tetrahedral Co(II) complexes are observed for both peptides in aqueous solution but with 4:2 peptide:Co(II) stoichiometries as estimated by ultracentrifugation. Cu(II) reduction potentials for the aqueous peptide-Cu(II) complexes were measured to be +75 +/- 30 mV vs NHE for BCP-A-Cu(II) and -10 +/- 20 mV vs NHE for BCP-B-Cu(II). The results indicate that the plastocyanin ligand loop can act as a metal-binding site with His and Cys ligands in the absence of the remainder of the folded protein but, by itself, cannot stabilize a type 1 copper site, emphasizing the role of the protein matrix in protecting the Cu binding site from solvent exposure and the Cys from oxidation.
Daugherty
Roxanne G
RG
Department of Chemistry, Texas A&M University, College Station, Texas 77842-3012, and Department of Chemistry, Columbia University, New York, New York 10027.
Wasowicz
Tomasz
T
Gibney
Brian R
BR
DeRose
Victoria J
VJ
D000465:Algorithms; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry*; D003035:Cobalt / Q000737:chemistry*; D003300:Copper / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D008958:Models, Molecular; D008968:Molecular Conformation; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry*; D010970:Plastocyanin / Q000737:chemistry*; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D002352:Carrier Proteins; D010455:Peptides; D003035:Cobalt; D003300:Copper; D010970:Plastocyanin