Homology predicted structure and functional interaction of ferredoxin from the eukaryotic alga Chlamydomonas reinhardtii with nitrite reductase and glutamate synthase.
Ferredoxin (Fd) from Chlamydomonas reinhardtii is composed of 94 amino-acid residues and a [2Fe-2S] cluster. The homology modelling technique has been used to predict the tertiary structure of C. reinhardtii Fd. The overall structure shows the typical fifth-stranded beta-grasp plus two additional beta-sheets and three alpha-helices. Site-directed mutagenesis of recombinant Fd has allowed us to obtain four point mutants and one double mutant--all mutations being located in the short alpha-helix at the carboxy-terminal segment as well as a triple mutant affected on helix alpha1. Crosslinking studies and measurement of enzymatic activities reveal that the residues changed are critical for the interaction of Fd with glutamate synthase (GOGAT) and nitrite reductase (NiR). Potentiometric analyses of the Fd mutants show that the replacement of glutamate in position 91 drastically changes the redox potential value (70 mV), thereby suggesting that such a glutamate can modulate the reactivity of Fd towards its reaction partners. According to results herein presented, the reported mutations modify the electrostatic interactions within the complex formed between Fd and GOGAT or NiR.
García-Sánchez
M I
MI
Instituto de Bioquímica Vegetal y Fotosíntesis, CSIC y Universidad de Sevilla, Centro de Investigaciones Científicas Isla de la Cartuja,  Spain.
Díaz-Quintana
A
A
Gotor
C
C
Jacquot
J P
JP
De la Rosa
M A
MA
Vega
J M
JM
D000595:Amino Acid Sequence; D000818:Animals; D016825:Chlamydomonas reinhardtii / Q000378:metabolism*; D005288:Ferredoxins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005970:Glutamate Synthase / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000378:metabolism*; D011485:Protein Binding; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity; D013329:Structure-Activity Relationship
D005288:Ferredoxins; D005970:Glutamate Synthase; D009565:Nitrate Reductases; D050901:Nitrate Reductase