Electron transfer to photosystem 1 from spinach plastocyanin mutated in the small acidic patch: Ionic strength dependence of kinetics and comparison of mechanistic models

A set of plastocyanin (Pc) mutants, probing the small acidic patch (Glu59, Glu60, and Asp61) and a nearby residue, Gln88, has been constructed to prov ide further insight into the electron transfer process between Pc and photo system 1. The negatively charged residues were changed into their neutral c ounterparts or to a positive lysine. All mutant proteins exhibited electron transfer kinetics qualitatively similar to those of the wild type protein over a wide range of Pc concentrations. The kinetics were slightly faster f or the Gln88Lys mutant, while they were significantly slower for the Glu59L ys mutant. The data were analyzed with two different models: one involving a conformational change of the Pc-photosystem 1 complex that precedes the e lectron transfer step (assumed to be irreversible) [Bottin, H., and Mathis, P. (1985) Biochemistry 24, 6453-6460] and another where no conformational change occurs, the electron transfer step is reversible, and dissociation o f products is explicitly taken into account [Drepper, F., Hippler, M., Nits chke, W., and Haehnel, W. (1996) Biochemistry 35, 1282-1295]. Both models c an account for the observed kinetics in the limits of low and high Pc conce ntrations. To discriminate between the models, the effects of added magnesi um ions on the kinetics were investigated. At a high Pc concentration (0.7 mM), the ionic strength dependence was found to be consistent with the mode l involving a conformational change but not with the model where the electr on transfer is reversible. One residue in the small acidic patch, Glu60, se ems to be responsible for the major part of the ionic strength dependence o f the kinetics.