Binding of Yeast Cytochrome <i>c</i> to Forty-Four Charge-Reversal Mutants of Yeast Cytochrome <i>c</i> Peroxidase: Isothermal Titration Calorimetry

Previously, we constructed, expressed, and purified 46 charge-reversal mutants of yeast cytochrome <i>c</i> peroxidase (CcP) and determined their electronic absorption spectra, their reaction with H₂O₂, and their steady-state catalytic properties [Pearl, N. M. et al. (2008) Biochemistry 47, 2766−2775]. Forty-four of the mutants involve the conversion of either an aspartate or glutamate residue to a lysine residue, while two are positive-to-negative mutations, R31E and K149D. In this paper, we report on a calorimetric study of the interaction of each charge-reversal mutant (excluding the internal mutants D76K and D235K) with recombinant yeast iso-1 ferricytochrome <i>c</i>(C102T) (yCc) under conditions where only one-to-one yCc/CcP complex formation is observed. Thirteen of the 44 surface-site charge-reversal mutants decrease the binding affinity for yCc by a factor of 2 or more. Eight of the 13 mutations (E32K, D33K, D34K, E35K, E118K, E201K, E290K, E291K) occur within, or on the immediate periphery, of the crystallographically defined yCc binding site [Pelletier, H. and Kraut, J. (1992) Science 258, 1748−1755], three of the mutations (D37K, E98K, E209K) are slightly removed from the crystallographic site, and two of the mutations (D165K, D241K) occur on the “back-side” of CcP. The current study is consistent with a model for yCc binding to CcP in which yCc binds predominantly near the region defined by crystallographic structure of the 1:1 yCc–CcP complex, whether as a stable electron-transfer active complex or as part of a dynamic encounter complex