| Abstract
| - Isothermal titration calorimetry was used to study the formation of 19 complexes involvingyeast iso-1-ferricytochrome c (Cc) and ferricytochrome c peroxidase (CcP). The complexes comprisedcombinations of the wild-type proteins, six CcP variants, and three Cc variants. Sixteen protein combinationswere designed to probe the crystallographically defined interface between Cc and CcP. The data showthat the high-affinity sites on Cc and CcP coincide with the crystallographically defined sites. Changingcharged residues to alanine increases the enthalpy of complex formation by a constant amount, but thedecrease in stability depends on the location of the amino acid substitution. Deleting methyl groups hasa small effect on the binding enthalpy and a larger deleterious effect on the binding free energy, consistentwith model studies of the hydrophobic effect, and showing that nonpolar interactions also stabilize thecomplex. Double-mutant cycles were used to determine the coupling energies for nine Cc−CcP residuepairs. Comparing these energies to the crystal structure of the complex leads to the conclusion that manyof the substitutions induce a rearrangement of the complex.
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