title: Mutations of Glu92 in ferredoxin I from spinach leaves produce proteins fully functional in electron transfer but less efficient in supporting NADP+ photoreduction.
abstract: Ferredoxin I in spinach chloroplasts fulfils the role of distributing electrons of low redox potential produced by photosystem I to several metabolic routes, NADP+ reduction being the major output. To investigate the role of Glu92, which is conserved in the chloroplast-type ferredoxins, mutations of this residue to either Gln, Ala or Lys were obtained through site-directed mutagenesis. A Glu93Ala mutant was also designed. The four mutants of ferredoxin I were overproduced in Escherichia coli, purified and characterised. The different migration in nondenaturing gel electrophoresis of wild-type and mutant proteins confirmed that the desired mutation was present in the expressed proteins. Spectral and physical properties of the mutants were similar to those of wild-type ferredoxin; electron-transfer properties were, however, quite different in the case of the mutants at position 92. Unexpectedly, these mutant ferredoxins were found to be twice as active as the wild-type protein in supporting the NADPH--cytochrome c reductase reaction catalysed by ferredoxin--NADP+ reductase. However, interactions of the mutant ferredoxins with the isolated thylakoid membranes deprived of endogenous ferredoxin showed that the mutants were less capable of supporting NADP+ photoreduction than the wild-type protein: both V and the apparent Km for reduced ferredoxin were influenced. On the other hand, the Kd values for the complex between oxidised ferredoxin and the reductase, measured at low ionic strength, were substantially changed only in the case of the Glu-->Lys mutation. With this mutant the rate of cross-linking between the two proteins induced by a carbodiimide was also decreased. It was found that the redox potentials of the iron-sulfur cluster of the mutants were more positive by 73-93 mV than that of ferredoxin I. Thus, the behavior of the ferredoxin mutants can be rationalised in terms of the effect of the side-chain replacement on the electrochemical properties of the [2Fe-2S] cluster and of an impairment in the interaction with the reductase under physiological conditions.
authors.affiliation: Dipartimento di Fisiologia e Biochimica Generali, Universitá degli Studi di Milano, Italy.
authors.forename: L
authors.initials: L
authors.lastname: Piubelli
authors.forename: A
authors.initials: A
authors.lastname: Aliverti
authors.forename: F
authors.initials: F
authors.lastname: Bellintani
authors.forename: G
authors.initials: G
authors.lastname: Zanetti
mesh_terms: D001483:Base Sequence; D002736:Chloroplasts / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004579:Electron Transport; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism*; D005288:Ferredoxins / Q000737:chemistry* / Q000378:metabolism; D005971:Glutamates / Q000737:chemistry; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D010084:Oxidation-Reduction; D010788:Photosynthesis; D011485:Protein Binding; D018724:Spinacia oleracea
chemical_list: D017931:DNA Primers; D005288:Ferredoxins; D005971:Glutamates; D007506:Iron-Sulfur Proteins; D009249:NADP; D005287:Ferredoxin-NADP Reductase