Previous studies, and the three-dimensional structure of Anabaena PCC 7119 ferredoxin-NADP+ reductase (FNR), indicate that the positive charge of Lys75 might be directly involved in the interaction between FNR and its protein partners, ferredoxin (Fd) and flavodoxin (Fld). To assess this possibility, this residue has been replaced by another positively charged residue, Arg, by two uncharged residues, Gln and Ser, and by a negatively charged residue, Glu. UV-vis absorption, fluorescence, and CD spectroscopies of these FNR mutants (Lys75Arg, Lys75Gln, Lys75Ser, and Lys75Glu) indicate that all the mutated proteins folded properly and that significant protein structural rearrangements did not occur. Steady-state kinetic parameters for these FNR mutants, utilizing the diaphorase activity with DCPIP, indicate that Lys75 is not a critical residue for complex formation and electron transfer (ET) between FNR and NADP+ or NADPH. However, steady-state kinetic activities requiring complex formation and ET between FNR and Fd or Fld were appreciably affected when the positive charge at position of Lys75 was removed, and the ET reaction was not even measurable if a negatively charged residue was placed at this position. These kinetic parameters also suggest that it is complex formation that is affected by mutation. Consistent with this, when dissociation constants (Kd) for FNRox-Fdox (differential spectroscopy) and FNRox-Fdrd (laser flash photolysis) were measured, it was found that neutralization of the positive charge at position 75 increased the Kd values by 50-100-fold, and that no complex formation could be detected upon introduction of a negative charge at this position. Fast transient kinetic studies also corroborated the fact that removal of the positive charge at position 75 of FNR appreciably affects the complex formation process with its protein partners 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 physiological protein partners. The results also suggest that the interaction of this residue with its protein partners is not structurally specific, since Lys75 can still be efficiently substituted by an arginine, but is definitely charge specific.