Nuclear magnetic resonance studies of the copper binding sites of blue copper proteins: oxidized, reduced, and apoplastocyanin.

Proton nuclear resonance spectra at 250 MHz of plastocyanins from spinach (Spinacia oleracea) and a blue green alga (Anabaena variabilis) are reported. Spectra of the reduced plastocyanins contain well-resolved peaks from slowly exchangeable N-H, histidine C2-H tyrosine ring, peptide alpha-CH, and high-field protons. The widths of these peaks indicate that the plastocyanins are monomeric. When the plastocyanins are oxidized, several changes in the spectra are observed including disappearance of peaks assigned to two histidine side chains. The pKa' values of the two histidine residues of reduced spinach plastocyanin are abnormally low (4.9 and less than 4.5). These pKa' values become more normal in apoplastocyanin or plastocyanin inhibited by cyanide. The results suggest that the imidazole groups of the two histidine residues are liganded directly to the copper in plastocyanin. The displacement of copper by cyanide is reversed at low pH. Spectra of apo- and reduced plastocyanins show only minor differences. However, the slowly exchangeable protons of plastocyanin exchange more rapidly in the apoprotein. Copper binding apparently does not cause a major reorganization of the protein structure, but the presence of copper does stabilize this structure.