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 mut ated by site directed mutagenesis, using PCR and the expression plasmi d pET-Fd as a template. The recombinant mutated proteins were purified to homogeneity and tested in the activation of NADP-malate dehydrogen ase, a light dependent reaction in which ferredoxin thioredoxin reduct ase (FTR) and thioredoxin are involved. The mutation of residue Glu-91 (E92 in spinach, E94 in Anabaena) alone, either to Gin (E91Q) or to L ys (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 additio n, a triple mutant (D25A/E28Q/E29Q) was also found to be inactive for this redox dependent light activation. All these mutations had much we aker effects on the ferredoxin/ferredoxin NADP reductase interaction a s measured by the cytochrome c reduction assay. These results indicate that there is a recognition site for FTR in the C terminus part of fe rredoxin, but also that a core of negatively charged residues in the a lpha 1 helix of ferredoxin might be important in the general process o f light activation.