Cytochrome c Peroxidase−Cytochrome c Complex:  Locating the Second Binding Domain on Cytochrome c Peroxidase with Site-Directed Mutagenesis

Cytochrome c peroxidase (CcP) can bind as many as two cytochrome c (Cc) molecules in an electrostatic complex. The location of the two binding domains on CcP has been probed by photoinduced interprotein electron transfer (ET) between zinc-substituted horse cytochrome c (ZnCc) and CcP with surface charge-reversal mutations and by isothermal titration calorimetry (ITC). These results, which are the first experimental evidence for the location of domain 2, indicate that the weak-binding domain includes residues 146−150 on CcP. CcP(E290K) has a charge-reversal mutation in the tight-binding domain, which should weaken binding, and it weakens the 1:1 complex; K 1 decreases 20-fold at 18 mM ionic strength. We have employed two mutations to probe the proposed location for the weak-binding domain on the CcP surface: (i) D148K, a “detrimental” mutation with a net (+2) change in the charge of CcP, and (ii) K149E, a “beneficial” mutation with a net (−2) change in the charge. The interactions between FeCc and CcP(WT and K149E) also have been studied with ITC. The CcP(D148K) mutation causes no substantial change in the 2:1 binding but an increase in the reactivity of the 2:1 complex. The latter can be interpreted as a long-range influence on the heme environment or, more likely, the enhancement of a minority subset of binding conformations with favorable pathways for ET. CcP(K149E) has a charge-reversal mutation in the weak-binding domain that produces a substantial increase in the 2:1 binding constant as measured by both quenching and ITC. For the 1:1 complex of CcP(WT), ΔG 1 = −8.2 kcal/mol (K 1 = 1.3 × 106 M-1), ΔH 1 = +2.7 kcal/mol, and ΔS 1 = +37 cal/K·mol at 293 K; for the second binding stage, K 2 < 5 × 103 M-1, but accurate thermodynamic parameters were not obtained. For the 1:1 complex of CcP(K149E), ΔG 1 = −8.5 kcal/mol (K 1 = 2 × 106 M-1), ΔH 1 = +2.0 kcal/mol, and ΔS 1 = +36 cal/K·mol; for the second stage, ΔG 2 = −5.5 kcal/mol (K 1 = 1.3 × 104 M-1), ΔH 2 = +2.9 kcal/mol, and ΔS 2 = +29 cal/K·mol.