Residue Glu-91 of Chlamydomonas reinhardtii ferredoxin is essential for electron transfer to ferredoxin-thioredoxin reductase.
The [2Fe-2S] soluble ferredoxin from Chlamydomonas reinhardtii was mutated by site directed mutagenesis, using PCR and the expression plasmid pET-Fd as a template. The recombinant mutated proteins were purified to homogeneity and tested in the activation of NADP-malate dehydrogenase, a light dependent reaction in which ferredoxin thioredoxin reductase (FTR) and thioredoxin are involved. The mutation of residue Glu-91 (E92 in spinach, E94 in Anabaena) alone, either to Gln (E91Q) or to Lys (E91K), was found to completely abolish the reaction of the enzyme light activation. On the other hand, the mutants (E92Q) or (E92K) were as efficient as the wild type ferredoxin in this reaction whereas the double mutants (E91Q/E92Q) or (E91K/E92K) had no activity. In addition, a triple mutant (D25A/E28Q/E29Q) was also found to be inactive for this redox dependent light activation. All these mutations had much weaker effects on the ferredoxin/ferredoxin NADP reductase interaction as measured by the cytochrome c reduction assay. These results indicate that there is a recognition site for FTR in the C terminus part of ferredoxin, but also that a core of negatively charged residues in the alpha1 helix of ferredoxin might be important in the general process of light activation.
Jacquot
J P
JP
URA 1128 CNRS, Université de Paris-Sud, Institut de Biotechnologie desPlantes, France. jacquot@ibp.u-psud.fr
Stein
M
M
Suzuki
A
A
Liottet
S
S
Sandoz
G
G
Miginiac-Maslow
M
M
D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000378:metabolism*; D004579:Electron Transport; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism; D005288:Ferredoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018698:Glutamic Acid / Q000737:chemistry; D007506:Iron-Sulfur Proteins; D008027:Light; D008291:Malate Dehydrogenase / Q000378:metabolism; D050538:Malate Dehydrogenase (NADP+); D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D005288:Ferredoxins; D007506:Iron-Sulfur Proteins; D011994:Recombinant Proteins; D018698:Glutamic Acid; D010088:Oxidoreductases; D008291:Malate Dehydrogenase; D050538:Malate Dehydrogenase (NADP+); C021588:ferredoxin-thioredoxin reductase; D005287:Ferredoxin-NADP Reductase