Electron transfer from plastocyanin to photosystem I.
Mutant plastocyanins with Leu at position 10, 90 or 83 (Gly, Ala and Tyr respectively in wildtype) were constructed by site-specific mutagenesis of the spinach gene, and expressed in transgenic potato plants under the control of the authentic plastocyanin promoter, as well as in Escherichia coli as truncated precursor intermediates carrying the C-terminal 22 amino acid residues of the transit peptide, i.e. the thylakoid-targeting domain that acts as a bacterial export signal. The identity of the purified plastocyanins was verified by matrix-assisted laser desorption/ionization mass spectrometry. The formation of a complex between authentic or mutant spinach plastocyanin and isolated photosystem I and the electron transfer has been studied from the biphasic reduction kinetics of P700+ after excitation with laser flashes. The formation of the complex was abolished by the bulky hydrophobic group of Leu at the respective position of G10 or A90 which are part of the conserved flat hydrophobic surface around the copper ligand H87. The rate of electron transfer decreased by both mutations to < 20% of that found with wildtype plastocyanin. We conclude that the conserved flat surface of plastocyanin represents one of two crucial structural elements for both the docking at photosystem I and the efficient electron transfer via H87 to P700+. The Y83L mutant exhibited faster electron transfer to P700+ than did authentic plastocyanin. This proves that Y83 is not involved in electron transfer to P700 and suggests that electron transfer from cytochrome f and to P700 follows different routes in the plastocyanin molecule. Plastocyanin (Y83L) expressed in either E. coli or potato exhibited different isoelectric points and binding constants to photosystem I indicative of differences in the folding of the protein. The structure of the binding site at photosystem I and the mechanism of electron transfer are discussed.
Haehnel
W
W
Lehrstuhl für Biochemie der Pflanzen, Albert-Ludwigs-Universität, Freiburg, Germany.
Jansen
T
T
Gause
K
K
Klösgen
R B
RB
Stahl
B
B
Michl
D
D
Huvermann
B
B
Karas
M
M
Herrmann
R G
RG
D000595:Amino Acid Sequence; D004579:Electron Transport; D004926:Escherichia coli; D017343:Genes, Plant; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry* / Q000378:metabolism*; D045331:Photosystem I Protein Complex; D030821:Plants, Genetically Modified; D010970:Plastocyanin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D011198:Solanum tuberosum / Q000378:metabolism; D014675:Vegetables / Q000378:metabolism
D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; D011994:Recombinant Proteins; D010970:Plastocyanin