Electron transfer from plastocyanin to photosystem I.

Abstract

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.

Dynamic Interaction of Plastocyanin with the Cytochrome bf Complex

“…In analogy, replacing amino acid residues with small side chains in the hydrophobic surface (Fig. 1) by larger ones should inhibit the binding and the electron transfer as found for the interaction of plastocyanin with PSI (12). The six mutations of plastocyanin (A90L, A90T, G10L, G10M, G10V, and L12A) exert a small but noticeable effect on the second-order rate constant of the electron transfer from the cyt bf complex.…”

“…Wild-type plastocyanin was purified from spinach leaves (33). Recombinant plastocyanin was obtained by expression of a truncated precursor gene in Escherichia coli as described (12) and purified from the medium by ion exchange chromatography. The mutations were verified by electrospray mass spectrometry of the proteins (9).…”

“…The mutations were verified by electrospray mass spectrometry of the proteins (9). The mutants G10L, A90L, and Y83L have been previously described (12). Fig.…”

“…The Hydrophobic Surface-The electron transfer via the copper ligand His-87 in the center of the hydrophobic surface of plastocyanin has been favored for PSI (12). A tight fit of the hydrophobic surfaces of plastocyanin and cyt f could contribute to the formation of the electron transfer complex as well.…”

“…One is the flat hydrophobic area around the solventexposed copper ligand His-87, and the other is composed of two conserved acidic patches, Asp-42/Glu-43/Asp-44/Glu-45 and Glu-59/Glu-60/Asp-61, both adjacent to Tyr-83. To investigate the role of these elements in the interaction with PSI, crosslinking between plastocyanin and PSI (8,9,16), as well as measurements of electron transfer kinetics with site-directed plastocyanin mutants (9,10,12,14), have been conducted. The results suggest that in the complex the electron transfer occurs via the copper ligand His-87 (10,12) and that, apart from the interaction of the hydrophobic area, only the negative patch of residues 42-45 contributes substantially an electrostatic attraction to a positive domain of the PsaF subunit of PSI in plants (9,13,14).…”