We have used microcalorimetry and analytical ultracentrifugation to test the model proposedin Pettigrew et al. [(1999)
J. Biol. Chem. 274, 11383-11389] for the binding of small cytochromes to thecytochrome
c peroxidase of
Paracoccus denitrificans. Both methods reveal complexity in behavior dueto the presence of a monomer/dimer equilibrium in the peroxidase. In the presence of either Ca
2+, orhigher ionic strength, this equilibrium is shifted to the dimer. Experiments to study complex formationwith redox partners were performed in the presence of Ca
2+ in order to simplify the equilibria that hadto be considered. The results of isothermal titration calorimetry reveal that the enzyme can bind twomolecules of horse cytochrome
c with
Kd values of 0.8
M and 2.5
M (at 25
C, pH 6.0,
I = 0.026) butonly one molecule of
Paracoccus cytochrome
c-550 with a
Kd of 2.8
M, molar binding ratios confirmedby ultracentrifugation. For both horse cytochrome
c and
Paracoccus cytochrome
c-550, the binding isendothermic and driven by a large entropy change, a pattern consistent with the expulsion of water moleculesfrom the interface. For horse cytochrome
c, the binding is weakened 3-fold at
I = 0.046 M due to asmaller entropy change, and this is associated with an increase in enzyme turnover. In contrast, neitherthe binding of cytochrome
c-550 nor its oxidation rate is affected by raising the ionic strength in thisrange. We propose that, at low ionic strength, horse cytochrome
c is trapped in a nonproductive orientationon a broad capture surface of the peroxidase.