| Cytochrome c peroxidase (CcP) can bind 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 C cP with surface charge-reversal mutations and by isothermal titration calorimetry (ITC). CcP(E290K) has a charge-reversal mutation in domain 1, and it weakens the 1:1 complex 20-fold. Two mutations have been employed to probe the proposed location for the weakly-binding domain on the CcP surface; 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 CcP(K149E) mutation produces a substantial increase in the 2:1 binding constant as measured both by quenching and ITC. These results, the first experimental evidence for the location of domain 2, indicate that the weakly-binding domain is near residues 146--150 on CcP.;ITC experiments at higher, millimolar concentrations for both iron(II) and iron(III) horse cytochrome c have yielded lower binding constants for both binding steps, particularly the second, than was obtained at lower concentrations; K1 is reduced two-fold at high concentration and K2 is reduced 10-fold, to 7(2) x 102 M -1. Electron transfer quenching measurements at millimolar concentrations performed by monitoring ZnCcP emission give a similarly lower binding constant, K2 = 1.0(5) x 103 M -1. The thermodynamic implications of the concentration dependence of the binding "constant" are discussed.;While the weakly-binding domain 2 is more reactive than domain 1 to direct heme-heme ET as measured between ZnCc and Fe3+C cP, the question of whether domain 2 is more reactive in a reaction where Trp 191 is involved has not previously been addressed. Thus, experiments in which 3ZnCc reacts with compound-I C cP (CcP-I) and the oxyferryl form of compound-II (CcP-IIh) have been performed. In cases where Trp 191 cannot react, either because it is not there, as in the mutant C cP(W191F), or because it cannot be oxidized (the Fe3+C cP → Fe3+CcP reaction), domain 2, which is closer to the heme, is more reactive than domain 1. In the reactions between ZnCc and CcP-I or CcP-II h, domain 1, which is closer to the Trp, is more reactive than domain 2. |
