AMINO-ACID-RESIDUES INVOLVED IN FUNCTIONAL INTERACTION OF VEGETATIVE CELL FERREDOXIN FROM THE CYANOBACTERIUM ANABAENA SP PCC-7120 WITH FERREDOXIN-NADP REDUCTASE, NITRITE REDUCTASE AND NITRATE REDUCTASE

During evolution, cyanobacterial [2Fe-2S]-ferredoxins have developed structural features that enable them to form 1:1 complexes with different redox partners, which are mainly stabilized by electrostatic interactions. Three important ferredoxin-binding proteins are ferredoxin:NADP reductase (FNR), nitrite reductase(NIR) and nitrate reductase (NAR). Using site-directed mutagenesis with recombinant ferredoxin from vegetative cells (PetF) of Anabaena 7120, we measured the in vitro activities of these enzymes in ferredoxin-dependent steady-state assays. Aside from a major contribution of surface charge, electron transfer from reduced ferredoxin to all three redox partners was severely inhibited by amino acid substitutions at positions E94 and F65. However, mutations at these and other sites affected rates of electron transport to FNR, NIR and NAR differently. This suggested a common, but not identical, ferredoxin binding-domain on FNR, NIR and NAR. Residues E94 and F65 were previously shown to be required for rapid electron transfer to FNR [Hurley et al. (1993) Biochemistry 32, 9346-9354]. We show here additionally that the reversed electron flow from NADPH-reduced FNR to oxidized ferredoxin was strongly stimulated by the mutated proteins E94Q and F65A that showed almost no electron transport capacity to oxidized FNR. This supports a conformational change of the FNR induced by NADPH-binding and subsequent electron transfer supporting dissociation of the FNR-ferredoxin complex.