Identification of the amino acids involved in the functional interaction between photosystem I and ferredoxin from Synechocystis sp. PCC 6803 by chemical cross-linking.
Ferredoxin isolated from the cyanobacterium Synechocystis sp. PCC 6803 has been chemically cross-linked to purified photosystem I from the same organism. The reaction was catalyzed by N-ethyl-3-(3-dimethylaminopropyl)carbodiimide in the presence of N-hydroxysulfosuccinimide. A short reaction time and neutral pH values can be used in the presence of the two reagents, ensuring the integrity of both of the proteins and the iron-sulfur cluster of the ferredoxin. The only covalent complex detected comprised ferredoxin and the photo-system I (PSI)-D subunit, as identified by antibodies probing after electrophoresis. Electron paramagnetic resonance measurements of this covalent complex have shown that the cross-linked ferredoxin was entirely photoreducible by photosystem I and that the molar ratio of ferredoxin to PSI was close to 1. Extensive sequencing of the peptides obtained after proteolysis of the purified cross-linked product led to the identification of a covalent bond between glutamic acid 93 of ferredoxin and lysine 106 of the PSI-D subunit.
Lelong
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Service de Bioénergétique/URA 1290 (CNRS), Département de Biologie Cellulaire et Moléculaire, C. E. A., Saclay, Gif sur Yvette, France.
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Lagoutte
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Bottin
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D000595:Amino Acid Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D000458:Cyanobacteria / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D005022:Ethyldimethylaminopropyl Carbodiimide; D005288:Ferredoxins / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D045322:Photosynthetic Reaction Center Complex Proteins / Q000302:isolation & purification / Q000378:metabolism*; D045331:Photosystem I Protein Complex
D003432:Cross-Linking Reagents; D005288:Ferredoxins; D010446:Peptide Fragments; D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; D005022:Ethyldimethylaminopropyl Carbodiimide