Changes in the reaction mechanism of electron transfer from plastocyanin to photosystem I in the cyanobacterium Synechocystis sp. PCC 6803 as induced by site-directed mutagenesis of the copper protein.
The kinetic mechanism of plastocyanin oxidation by photosystem I in the cyanobacterium Synechocystis sp. PCC 6803 is drastically changed by modifying the metalloprotein by site-directed mutagenesis. The mutations herein considered concern four specific residues, two in the east face and the other two in the hydrophobic patch of plastocyanin. The first set of mutants include D44A, D44K, D47A, and D47R, as well as the double mutants D44A/D47A and D44R/D47R; the second set consists of L12A and K33E. The kinetic efficiency of all these mutant plastocyanins has been analyzed by laser-flash absorption spectroscopy. The plastocyanin concentration dependence of the observed electron transfer rate constant (kobs) is linear with most mutant plastocyanins, as with wild-type plastocyanin, but exhibits a saturation plateau at high protein concentration with the double mutant D44R/D47R, which suggests the formation of a plastocyanin-PSI transient complex. The effect of ionic strength on kobs varies from the wild-type plastocyanin to some of the mutants, for instance D44K, for which the salt concentration dependence of kobs is just the reverse as compared to the wild-type protein. The ionic strength dependence of kobs with D44R/D47R exhibits a bell-shaped profile, which is similar to that of green algae and higher plants. These findings indicate that the double mutant D44R/D47R follows a reaction mechanism involving not only complex formation with PSI but also further reorientation to properly accommodate the redox centers prior to electron transfer, as is the case in most evolved species, whereas the wild-type copper protein reacts with PSI by following a simple collisional kinetic model.
De la Cerda
B
B
Instituto de Bioquímica Vegetal y Fotosíntesis, Centro "Isla de la Cartuja", Universidad de Sevilla y CSIC, Sevilla, Spain.
Navarro
J A
JA
Hervás
M
M
De la Rosa
M A
MA
D000458:Cyanobacteria / Q000737:chemistry* / Q000378:metabolism; D004579:Electron Transport; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000378:metabolism; D010970:Plastocyanin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013057:Spectrum Analysis / Q000379:methods; D013329:Structure-Activity Relationship
D045322:Photosynthetic Reaction Center Complex Proteins; D010970:Plastocyanin