Flavodoxin (Fld) has been demonstrated to bind to ferredoxin-NADP+ reductase A (FprA) in Pseudomonas putida. Tworesidues (Phe256, Lys259) of FprA are likely to be important forinteracting with Fld based on homology modeling. Sitedirectedmutagenesis and pH-dependent enzyme kinetics wereperformed to further examine the role of these residues. Thecatalytic efficiencies of FprA-Ala259 and FprA-Asp259 proteinswere two-fold lower than those of the wild-type FprA.Homology modeling also strongly suggested that these tworesidues are important for electron transfer. Thermodynamicproperties such as entropy, enthalpy, and heat capacitychanges of FprA-Ala259 and FprA-Asp259 were examined byisothermal titration calorimetry. We demonstrated, for the firsttime, that Phe256 and Lys259 are critical residues for theinteraction between FprA and Fld. Van der Waals interactionsand hydrogen bonding were also more important than ionicinteractions for forming the FprA-Fld complex.