Proton linkage in formation of the cytochrome c-cytochrome c peroxidase complex: electrostatic properties of the high- and low-affinity cytochrome binding sites on the peroxidase.
The electrostatic character of cytochrome c-cytochrome c peroxidase complex formation has been studied by potentiometric titration between pH 5.5 and 7.75. Potentiometric data obtained at ionic strength > or = 100 mM were adequately analyzed in terms of 1:1 complex formation while the simplest model capable of fitting similar data obtained at lower ionic strength involves the assumption of two inequivalent binding sites for the cytochrome on the peroxidase. The stability of cytochrome c binding at the high-affinity site is ca. three orders of magnitude greater than that observed for the low-affinity site and is optimal between pH 6.75 and 7. The electrostatic properties of the two binding sites are distinctly different because, at most values of pH, binding of cytochrome c to the high-affinity site results in proton release while binding of the cytochrome to the low-affinity site results in proton uptake. Furthermore, binding of the cytochrome to the low-affinity site appears to be least stable in the pH range where binding to the high-affinity site is optimal. Interestingly, the binding parameters derived from these measurements were independent of temperature, consistent with a substantial entropic contribution to complex stability. Ferricytochrome c binds to the peroxidase with a slightly greater affinity than does ferrocytochrome c, and no evidence for specific anion effects on complex stability was observed. At low ionic strength (< or = 50 mM) and high pH (7.75), the interaction of the two proteins is more complex and cannot be adequately analyzed in terms of the two-site model.
Mauk
M R
MR
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
Ferrer
J C
JC
Mauk
A G
AG
D001665:Binding Sites; D003574:Cytochrome c Group / Q000378:metabolism*; D003578:Cytochrome-c Peroxidase / Q000378:metabolism*; D004563:Electrochemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D009994:Osmolar Concentration; D011199:Potentiometry; D011485:Protein Binding; D011522:Protons
D003574:Cytochrome c Group; D011522:Protons; D003578:Cytochrome-c Peroxidase