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 provide further insight into the electron transfer process between Pc and photosystem 1. The negatively charged residues were changed into their neutral counterparts 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 for the Gln88Lys mutant, while they were significantly slower for the Glu59Lys mutant. The data were analyzed with two different models: one involving a conformational change of the Pc-photosystem 1 complex that precedes the electron 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 of products is explicitly taken into account [Drepper, F., Hippler, M., Nitschke, W., and Haehnel, W. (1996) Biochemistry 35, 1282-1295]. Both models can account for the observed kinetics in the limits of low and high Pc concentrations. To discriminate between the models, the effects of added magnesium 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 model involving a conformational change but not with the model where the electron transfer is reversible. One residue in the small acidic patch, Glu60, seems to be responsible for the major part of the ionic strength dependence of the kinetics.
Olesen
K
K
Biochemistry and Biophysics, Department of Chemistry, Göteborg University, Sweden.
Ejdebäck
M
M
Crnogorac
M M
MM
Kostić
N M
NM
Hansson
O
O
D004305:Dose-Response Relationship, Drug; D004579:Electron Transport; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009994:Osmolar Concentration; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000378:metabolism; D010970:Plastocyanin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D018724:Spinacia oleracea
D045322:Photosynthetic Reaction Center Complex Proteins; D011994:Recombinant Proteins; D010970:Plastocyanin