PROTON LINKAGE IN FORMATION OF THE CYTOCHROME-C CYTOCHROME-C-PEROXIDASE COMPLEX - ELECTROSTATIC PROPERTIES OF THE HIGH-AFFINITY AND LOW-AFFINITY CYTOCHROME BINDING-SITES ON THE PEROXIDASE

The electrostatic character of cytochrome c-cytochrome c peroxidase co mplex formation has been studied by potentiometric titration between p H 5.5 and 7.75. Potentiometric data obtained at ionic strength greater than or equal to 100 mM were adequately analyzed in terms of 1:1 comp lex formation while the simplest model capable of fitting similar data obtained at lower ionic strength involves the assumption of two inequ ivalent binding sites for the cytochrome on the peroxidase. The stabil ity of cytochrome c binding at the high-affinity site is ca. three ord ers 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 p roton release while binding of the cytochrome to the low-affinity site results in proton uptake. Furthermore, binding of the cytochrome to t he low-affinity site appears to be least stable in the pH range where binding to the high-affinity site is optimal. Interestingly, the bindi ng parameters derived from these measurements were independent of temp erature, consistent with a substantial entropic contribution to comple x stability. Ferricytochrome c binds to the peroxidase with a slightly greater affinity than does ferrocytochrome c, and no evidence for spe cific anion effects on complex stability was observed. At low ionic st rength (less than or equal to 50 mM) and high pH (7.75), the interacti on of the two proteins is more complex and cannot be adequately analyz ed in terms of the two-site model.