Side-chain interactions in the plastocyanin-cytochrome f complex

Cytochrome f and plastocyanin are redox partners in the photosynthetic elec tron-transfer chain. Electron transfer from cytochrome f to plastocyanin oc curs in a specific short-lived complex. To obtain detailed information abou t the binding interface in this transient complex, the effects of binding o n the backbone and side-chain protons of plastocyanin have been analyzed by mapping NMR chemical-shift changes. Cytochrome f was added to plastocyanin up to 0.3 M equiv, and the plastocyanin proton chemical shifts were measur ed. Out of approximately 500 proton resonances, 86% could be observed with this method. Nineteen percent demonstrate significant chemical-shift change s and these protons are located in the hydrophobic patch (including the cop per ligands) and the acidic patches of plastocyanin, demonstrating that bot h areas are part of the interface in the complex. This is consistent with t he recently determined structure of the complex [Ubbink, M., Ejdeback, M., Karlsson, B. G., and Bendall, D. S, (1998) Structure 6, 323-335]. The large st chemical-shift changes are found around His87 in the hydrophobic patch, which indicates tight contacts and possibly water exclusion from this part of the protein interface. These results support the idea that electron tran sfer occurs via His87 to the copper in plastocyanin and suggest that the hy drophobic patch determines the specificity of the binding. The chemical-shi ft changes in the acidic patches are significant but small, suggesting that the acidic groups are involved in electrostatic interactions but remain so lvent exposed. The existence of small differences between the present data and those used for the structure may imply that the redox state of the meta ls in both proteins slightly affects the structure of the complex. The chem ical-shift mapping is performed on unlabeled proteins, making it an efficie nt way to analyze effects of mutations on the structure of the complex.