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 Ca(2+), 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 Ca(2+) 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 microM and 2.5 microM (at 25 degrees C, pH 6.0, I = 0.026) but only one molecule of Paracoccus cytochrome c-550 with a K(d) of 2.8 microM, molar binding ratios confirmed by ultracentrifugation. For both horse cytochrome c and Paracoccus cytochrome c-550, the binding is endothermic 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.
Pettigrew
Graham W
GW
Department of Preclinical Veterinary Sciences, Royal (Dick) School of Veterinary Studies, University of Edinburgh, Summerhall, Edinburgh EH9 1QH, UK. g.pettigrew@ed.ac.uk
Goodhew
Celia F
CF
Cooper
Alan
A
Nutley
Margaret
M
Jumel
Kornelia
K
Harding
Stephen E
SE
D000818:Animals; D002151:Calorimetry; D002152:Calorimetry, Differential Scanning; D003574:Cytochrome c Group / Q000737:chemistry; D003578:Cytochrome-c Peroxidase / Q000737:chemistry*; D019281:Dimerization; D004579:Electron Transport; D006736:Horses; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000737:chemistry; D010231:Paracoccus denitrificans / Q000201:enzymology*; D011485:Protein Binding; D017186:Titrimetry; D014461:Ultracentrifugation
D003574:Cytochrome c Group; D046911:Macromolecular Substances; D008565:Membrane Proteins; C031732:cytochrome C-550; D003578:Cytochrome-c Peroxidase