LYS75 OF ANABAENA FERREDOXIN-NADP(-TRANSFER() REDUCTASE IS A CRITICALRESIDUE FOR BINDING FERREDOXIN AND FLAVODOXIN DURING ELECTRON)

Previous studies, and the three-dimensional structure of Anabaena PCC 7119 ferredoxin-NADP(+) reductase (FNR), indicate that the positive ch arge of Lys75 might be directly involved in the interaction between FN R and its protein partners, ferredoxin (Fd) and flavodoxin (Fld). To a ssess this possibility, this residue has been replaced by another posi tively charged residue, Arg, by two uncharged residues, Gin and Ser, a nd by a negatively charged residue, Glu, UV-vis absorption, fluorescen ce, and CD spectroscopies of these FNR mutants (Lys75Arg, Lys75Gln, Ly s75Ser, and Lys75Glu) indicate that ail the mutated proteins folded pr operly and that significant protein structural rearrangements did not occur. Steady-state kinetic parameters for these FNR mutants, utilizin g the diaphorase activity with DCPIP, indicate that Lys75 is not a cri tical residue for complex formation and electron transfer (ET) between FNR and NADP(+) or NADPH. However, steady-state kinetic activities re quiring complex formation and Ep between FNR and Fd or FId were apprec iably affected when the positive charge at position of Lys75 was remov ed, and the ET reaction was not even measurable if a negatively charge d residue was placed at this position. These kinetic parameters also s uggest that it is complex formation that is affected by mutation. Cons istent with this, when dissociation constants (K-d) for FNRox-Fd(ox) ( differential spectroscopy) and FNRox-Fd(rd) (laser flash photolysis) w ere measured, it was found that neutralization of the positive charge at position 75 increased the K-d values by 50-100-fold, and that no co mplex formation could be detected upon introduction of a negative char ge at this position. Fast transient kinetic studies also corroborated the fact that removal of the positive charge at position 75 of FNR app reciably affects the complex formation process with its protein partne rs but indicates that ET is still achieved in all the reactions. This study thus clearly establishes the requirement of a positive charge at position Lys75 for complex formation during ET between FNR and its ph ysiological protein partners. The results also suggest that the intera ction of this residue with its protein partners is not structurally sp ecific, since Lys75 can still be efficiently substituted by an arginin e, but is definitely charge specific.