Residue Glu‐91 of Chlamydomonas reinhardtii ferredoxin is essential for electron transfer to ferredoxin‐thioredoxin reductase

FEBS Letters

Miginiac-Maslow, Myroslawa¹  Stein, Mariana¹  Liottet, Sandrine¹  Sandoz, Gabriel¹  Suzuki, Akira²  Jacquot, Jean-Pierre¹ 

[1]URA 1128 CNRS, Université de Paris-Sud, Institut de Biotechnologie des Plantes, Bâtiment 630, 91405 Orsay-Cedex, France

[2]Laboratoire d'étude du métabolisme, INRA, route de Saint-Cyr, 78000 Versailles Cedex, France

关键词: Chlamydomonas reinhardtii;    Ferredoxin;    Mutagenesis;    FNR;    ferredoxin NADP oxidoreductase;    FTR;    ferredoxin thioredoxin-reductase;    NADP-MDH;    NADP malate dehydrogenase;    WT;    wild type;   

DOI  :  10.1016/S0014-5793(96)01407-X

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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 α1 helix of ferredoxin might be important in the general process of light activation.

【 授权许可】

Unknown