ANALYSIS OF THE H-1-NMR CHEMICAL-SHIFTS OF CU(I)-SUBSTITUTED, CU(II)-SUBSTITUTED 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 H-1-NMR spectra of CuI-, CuII- and Cd-plastocyanin from pea have b een analyzed. Full assignments of the spectra of CuI- and Cd-plastocya nin indicate chemical shift differences up to 1 ppm The affected proto ns are located in the four loops;hat 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 ch anges in the network upon replacement of CuI by Cd. For CuII-plastocya nin the resonances of 72% of the protons observed in the CuI form have been identified. Protons within approximate to 0.9 nm of the CuII wer e not observed due to fast paramagnetic relaxation. The protons betwee n 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 c orrected for the pseudocontact shift contribution, the CuII-plastocyan in 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 co rrection. The fact that pseudocontact shifts were observed for protons which were not broadened may be attributable to the weaker distance d ependence of the pseudocontact shift effect compared to paramagnetic r elaxation. This results in two shells around the Cu atom, an inner par amagnetic shell (0-0.9 nm), in which protons are not observed due to b roadening, and an outer paramagnetic shell (0.9-1.7 nm). in which prot ons can be observed and show pseudocontact shifts. It is concluded tha t Cd-plastocyanin is a suitable redox-inactive substitute for Cu-plast ocyanin.