Biochemical characterization of ferredoxin-NADP⁺reductase interaction with flavodoxin in Pseudomonas putida

Flavodoxin (Fld) has been demonstrated to bind to ferredoxin-NADP⁺ reductase A (FprA) in Pseudomonas putida. Two residues (Phe²⁵⁶, Lys²⁵⁹) of FprA are likely to be important for interacting with Fld based on homology modeling. Sitedirected mutagenesis and pH-dependent enzyme kinetics were performed to further examine the role of these residues. The catalytic efficiencies of FprA-Ala²⁵⁹ and FprA-Asp²⁵⁹ proteins were two-fold lower than those of the wild-type FprA. Homology modeling also strongly suggested that these two residues are important for electron transfer. Thermodynamic properties such as entropy, enthalpy, and heat capacity changes of FprA-Ala²⁵⁹ and FprA-Asp²⁵⁹ were examined by isothermal titration calorimetry. We demonstrated, for the first time, that Phe²⁵⁶ and Lys²⁵⁹ are critical residues for the interaction between FprA and Fld. Van der Waals interactions and hydrogen bonding were also more important than ionic interactions for forming the FprA-Fld complex.