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., & 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 Cp, of -680 +/- 44 J mol-1 K-1. The favorable binding entropy and the negative delta Cp 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.
Jelesarov
I
I
Biochemisches Institut der Universität Zürich, Switzerland.
Bosshard
H R
HR
D002152:Calorimetry, Differential Scanning; D002736:Chloroplasts / Q000737:chemistry; D005288:Ferredoxins / Q000737:chemistry*; D010088:Oxidoreductases / Q000737:chemistry*; D011485:Protein Binding; D018724:Spinacia oleracea; D013816:Thermodynamics; D014867:Water / Q000737:chemistry
D005288:Ferredoxins; D014867:Water; D010088:Oxidoreductases; C028878:ferredoxin-NAD+ reductase