A spectroscopic and voltammetric study of the pH-dependent Cu(II) coordination to the peptide GGGTH:: relevance to the fifth Cu(II) site in the prion protein

The GGGTH sequence has been proposed to be the minimal sequence involved in the binding of a fifth Cu(II) ion in addition to the octarepeat region of the prion protein (PrP) which binds four Cu(II) ions. Coordination of Cu(II) by the N- and C-protected Ac-GGGTH-NH2 pentapeptide (P-5) was investigated by using potentiometric titration, electrospray ionization mass spectrometry, UV-vis spectroscopy, electron paramagnetic resonance (EPR) spectroscopy and cyclic voltammetry experiments. Four different Cu(H) complexes were identified and characterized as a function of pH. The Cu(II) binding mode switches from NO3 to N-4 for pH values ranging from 6.0 to 10.0. Quasi-reversible reduction of the [Cu-II(P-5)H-2] complex formed at pH 6.7 occurs at E-1/2=0. 04 V versus Ag/AgCl, whereas reversible oxidation of the [Cu-II(P-5)H-3](-) complex formed at pH 10.0 occurs at E-1/2=0.66 V versus Ag/AgCl. Comparison of our EPR data with those of the rSHaPrP(90-231) (Burns et al. in Biochemistry 42:6794-6803, 2003) strongly suggests an N3O binding mode at physiological pH for the fifth Cu(II) site in the protein.