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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 X 10(6) M-1. Though the enthalpy (delta H = 19 +/- 1 kJ.mol-1) and the entropy (delta S = 183 J.K-1.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 from 7.35 to 6.05.

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Thermodynamic data are reported for magnesium complex formation of GTP, ITP, UTP, GDP, UDP, GMP, and UMP. The AG, AH, and AS values were determined at 30°, pH 8.50, and µ = 0.2 with a microcalorimetric technique using an isothermic Calvet apparatus. In all cases the AH values are proportional to the length of the phosphate chains. On the other hand, the AG and AS values are dependent on the nature of the ring moiety, especially in the case of the nucleotide triphosphates. The affinity of the nucleotide triphosphates for magnesium increases in the order ATP > UTP > ITP, GTP. Moreover, the pÁT's of the rings of magnesiumcomplexed species are shifted to lower pH values. These facts are in good agreement with the hypothesis that the ring moiety of the nucleotides interacts weakly with Mg2+.The adenine nucleotide-magnesium complexes, because of their importance in a great many biological reactions, have been extensively studied. Much work has been done on the determination of the stability constant and thermodynamic quantities associated with complex formation. Unfortunately, research concerning the magnesium chelates of the other 5' ribonucleotides is very rare.The only work reported in this field is that of Walaas,1 2 whose results show no change in the stability constant for either magnesium or manganese ions, if uridine or guanosine triphosphate replaces ATP. Since that time, it has been routinely assumed that the stability constants are the same for all the homologous nucleotides; however, the values presently used, e.g., by Shramm and Morrison,3 are not those measured by Walaas, but those recently obtained for the formation of the adenine nucleotide-Mg2+ complexes.In this study, the thermodynamic quantities (AG, AH, AS) of magnesium complex formation of GTP, UTP, ITP, GDP, UDP, GMP, and UMP have been estimated by a microcalorimetric technique. (The usual abbreviations of adenosine, guanosine, uridine, and inosine mono-, di-, and triphosphate are used when there is no need to specify the ionic species precisely.) Experimental SectionApparatus. The experimental procedure and the microcalorimeter used are the same as those previously described by Belaich and Sari.4 However, for the mixing of reagents, the experimental "siphon cell" previously described is replaced by a cell which is equipped with a closing device (Figure 1). The opening and the mixing artefacts observed when 1 ml of buffer contained in this cell is injected into the Pyrex calorimetric cell containing 5 ml of the same buffer are less than 3 X 10~4 cal.