Analysis of the 1H-NMR chemical shifts of Cu(I)-, Cu(II)- and Cd-substituted pea plastocyanin. Metal-dependent differences in the hydrogen-bond network around the copper site.

To compare cadmium-substituted plastocyanin with copper plastocyanin, the 1H-NMR spectra of CuI-, CuII- and Cd-plastocyanin from pea have been analyzed. Full assignments of the spectra of CuI- and Cd-plastocyanin indicate chemical shift differences up to 1 ppm. The affected protons are located in the four loops that surround the Cu site. The largest differences were found for protons in the hydrogen bond network which stabilizes this part of the protein. This suggests that the chemical shift differences are caused by very small but extensive structural changes in the network upon replacement of CuI by Cd. For CuII-plastocyanin the resonances of 72% of the protons observed in the CuI form have been identified. Protons within approximately 0.9 nm of the CuII were not observed due to fast paramagnetic relaxation. The protons between 0.9-1.7 nm from the CuII showed chemical shift differences up to 0.4 ppm compared to both CuI- and Cd-plastocyanin. These differences can be predicted assuming that they represent pseudocontact shifts. When corrected for the pseudocontact shift contribution, the CuII-plastocyanin chemical shifts were nearly all identical within error to those of the Cd form, but not of the CuI-plastocyanin, indicating that the CuII-plastocyanin structure, in as far as it can be observed, resembles Cd-rather than CuI-plastocyanin. In a single stretch of residues (64-69) chemical shift differences remained between all three forms after correction. The fact that pseudocontact shifts were observed for protons which were not broadened may be attributable to the weaker distance dependence of the pseudocontact shift effect compared to paramagnetic relaxation. This results in two shells around the Cu atom, an inner paramagnetic shell (0-0.9 nm), in which protons are not observed due to broadening, and an outer paramagnetic shell (0.9-1.7 nm), in which protons can be observed and show pseudocontact shifts. It is concluded that Cd-plastocyanin is a suitable redox-inactive substitute for Cu-plastocyanin.