The electron transfer complexes of cytochrome c peroxidase from Paracoccus denitrificans

We have used microcalorimetry and analytical ultracentrifugation to test the model proposed in Pettigrew et al. [(1999) J. Biol. Chem. 274, 11383-11389] for the binding of small cytochromes to the cytochrome c peroxidase of Paracoccus denitrificans. Both methods reveal complexity in behavior due to the presence of a monomer/dimer equilibrium in the peroxidase. In the presence of either Ca2+ or higher ionic strength, this equilibrium is shifted to the dimer. Experiments to study complex formation with redox partners were performed in the presence of Ca2+ in order to simplify the equilibria that had to be considered. The results of isothermal titration calorimetry reveal that the enzyme can bind two molecules of horse cytochrome c with K-d values of 0.8 muM and 2.5 muM (at 25 degreesC, pH 6.0, I = 0.026) but only one molecule of Paracoccus cytochrome c-550 with a K-d of 2.8 muM, molar binding ratios confirmed by ultracentrifugation. For both horse cytochrome c and Paracoccus cytochrome c-550, the binding is endothennic and driven by a large entropy change, a pattern consistent with the expulsion of water molecules from the interface. For horse cytochrome c, the binding is weakened 3-fold at I = 0.046 M due to a smaller entropy change, and this is associated with an increase in enzyme turnover. In contrast, neither the binding of cytochrome c-550 nor its oxidation rate is affected by raising the ionic strength in this range. We propose that, at low ionic strength, horse cytochrome c is trapped in a nonproductive orientation on a broad capture surface of the peroxidase.