On the role of the acidic cluster Glu 92-94 of spinach ferredoxin I.
The role of the acidic cluster Glu 92-94 of spinach ferredoxin I in the interaction both with the photosystem I multisubunit complex and the ferredoxin-NADP+ reductase, either membrane-bound or purified, was studied by kinetic characterization of site-directed mutants. Three mutants of ferredoxin have been produced to evaluate the effects of elimination of one or two negative charges in the three specific positions of the acidic cluster. Kinetic characterization of the ferredoxin mutants E92A/E93A, E93A and E93A/E94A as electron carriers in the photosynthetic electron transport chain, allowed to establish that the two latter mutants were nearly indistinguishable from the wild-type protein in their ability to be photoreduced by photosystem I and as electron donor to the reductase in the NADP+ photoreduction with thylakoid membranes. The E92A/E93A ferredoxin mutant behaved very similarly to E92 mutants previously characterized. Thus, the elimination of the carboxyl groups adjacent to residue 92 did not further impaired ferredoxin I main function, i.e., as an electron carrier in NADP+ photoreduction. The two double mutants showed a reduced rate in the cross-linking of ferredoxin to the reductase promoted by a soluble carbodiimide, indicating an involvement of the acidic cluster in the formation of the active covalent complex between the two proteins.
Aliverti
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Dipartimento di Fisiologia e Biochimica Generali, Università degli Studi di Milano, Milan, Italy.
Livraghi
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Piubelli
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Zanetti
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D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D003574:Cytochrome c Group / Q000378:metabolism; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism; D005288:Ferredoxins / Q000737:chemistry* / Q000378:metabolism*; D018698:Glutamic Acid; D007700:Kinetics; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010084:Oxidation-Reduction; D045322:Photosynthetic Reaction Center Complex Proteins / Q000378:metabolism; D045331:Photosystem I Protein Complex; D018724:Spinacia oleracea / Q000378:metabolism*
D003574:Cytochrome c Group; D005288:Ferredoxins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D045322:Photosynthetic Reaction Center Complex Proteins; D045331:Photosystem I Protein Complex; D018698:Glutamic Acid; D005287:Ferredoxin-NADP Reductase