THERMODYNAMICS OF FERREDOXIN BINDING TO FERREDOXIN-NADP(+) REDUCTASE AND THE ROLE OF WATER AT THE COMPLEX INTERFACE

The association of ferredoxin with ferredoxin:NADP(+) reductase (both proteins from spinach chloroplasts) was characterized by isothermal titration calorimetry and fluorescence quenching titration. The formation of the complex is mainly driven by a positive entropy change (Delta S = 125 +/- 8 J mol(-1) K-1). The calorimetric enthalpy of binding is small between 10 and 37 degrees C and either negative or positive, with an inversion temperature near 25 degrees C. The pH dependence of the association constant [Batie, C. J., and Kamin, H. (1981) J. Biol. Chem. 256, 7756-7763] was shown to correlate with the uptake of a single proton by a group exhibiting a heat of protonation of -26 kJ mol(-1). This value agrees with the protonation of an imidazole group. Possible residues to become protonated in the complex are His-19 or His-90 of ferredoxin:NADP(+) reductase. The temperature dependence of the free energy of binding, Delta G, is weak because of the enthalpy-entropy compensation caused by a heat capacity change, Delta C-p, of -680 +/- 44 J mol(-1) K-1. The favorable binding entropy and the negative Delta C-p indicate a large contribution to binding from hydrophobic effects, which seem to originate from dehydration of the protein-protein interface. Dehydration was demonstrated by osmotic stress experiments in which the association constant was found to increase by 2-4-fold in the presence of 52% (W/W) glycerol. The increase in the association constant with osmotic pressure points to the release of several water molecules from the complex interface.