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 cytocthrome 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; K₁ 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), ΔG₁ = -8.2 kcal/mol (K₁ = 1.3 x 10⁶ M^-1), ΔH₁ = +2.7 kcal/mol, and ΔS₁ = +37 cal/K·mol at 293 K; for the second binding stage, K₂ < 5 x 10³ M^-1, but accurate thermodynamic parameters were not obtained. For the 1:1 complex of CcP-(K149E), ΔG₁ = -8.5 kcal/mol (K₁ = 2 x 10⁶ M^-1), ΔH₁ = +2.0 kcal/mol, and ΔS₁ = +36 cal/K·mol; for the second stage, ΔG₂ = -5.5 kcal/mol (K₁ = 1.3 x 10⁴ M^-1), ΔH₂ = +2.9 kcal/mol, and ΔS₂ = +29 cal/K·mol.