Thermodynamic parameters of the cytochrome c3-ferredoxin complex formation

Abstract

The complex formation between cytochrome c3 and ferredoxin I from Desulfovibrio desulfuricans Norway was studied by microcalorimetric and pH-stat titration measurements. The stoichiometry of the complex was found to be one molecule of cytochrome c3 per monomer of ferredoxin I. The association constant determined at T = 283 K in tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) buffer, 10-2 M and pH 7.7, was KA = 1.3 .times. 106 M-1. Though the enthalpy (.DELTA.H = 19 .+-. 1 kJ .cntdot. mol-1) and the entropy (.DELTA.S = 183 J .cntdot. K-1 .cntdot. mol-1) were positive and consistent with a hydrophobic process involved in the interaction, the analysis of ionic strength dependence exhibited an important electrostatic effect on the association. The use of both Tris-HCl and phosphate buffers during microcalorimetric experiments showed proton release at pH 6.6. The pH-stat study of proton release indicated that one of the charged groups involved in the interacting site underwent a pK shift 7.35 to 6.05.