NMR and kinetic characterization of the interaction between French bean plastocyanin and horse cytochrome c.

French bean plastocyanin is shown by stopped-flow kinetics to oxidize horse cytochrome c with k (298 K, I = 0.10 M) = 5.1 X 10(6) M-1 X s-1. The activation parameters demonstrate a satisfactory isokinetic correlation with those previously reported for plastocyanin-cytochrome f reactions. NMR line broadening and shifts of the hyperfine shifted resonances of cytochrome c(III) reveal that strong 1:1 complexes are formed with plastocyanin. The negative patch of plastocyanin and the heme edge region of cytochrome c are shown to be the interacting sites by the hyperfine shift perturbations and competitive binding experiments with Gd3+, which associates selectively with the negative patch of plastocyanin. Complexation of plastocyanin and cytochrome c causes a small change in the heme electronic structure, but there is no NMR or optical evidence for significant conformational changes at either metal center. The rate of the reverse electron-transfer reaction within the plastocyanin-cytochrome c complex has been directly measured by NMR line broadening (krev (298 K) = 87 s-1). A rate for the forward intracomplex electron-transfer reaction (kf (298 K) = 4.8 X 10(3) s-1) has been calculated from krev and the optically measured equilibrium constant.