| Abstract
| - 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 Ca2+, orhigher ionic strength, this equilibrium is shifted to the dimer. Experiments to study complex formationwith redox partners were performed in the presence of Ca2+ 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 μM and 2.5 μM (at 25 °C, pH 6.0, I = 0.026) butonly one molecule of Paracoccus cytochrome c-550 with a Kd of 2.8 μM, 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.
|
