We have demonstrated that cytochrome c (Cc) binds to cytochrome c peroxidase (CcP) with a 2:1 stoichiometry, and that: (i) Cc reacts at two distinct and non-exclusive surface domains of CcP; (ii) two molecules of Cc can bind simultaneously to CcP; and (iii) the ternary complex is more reactive than the binary complex for the heme-heme reaction. The quenching studies, however, provide only stoichiometric (thermodynamic) parameters. They do not directly probe the microscopic properties of the individual domains, and thus do not apportion the reactivity due to 1:1 stoichiometry between the two types of binary complexes: one with Cc bound at the high-affinity domain and one with Cc bound at a low-affinity domain. We describe here a method for analyzing the stoichiometric parameters obtained from triplet quenching titrations to partition the quenching of ZnCc by CcP into contributions from the binary and ternary complexes, and also to give limits on the partition of the quenching into contributions from the low-and high-affinity binding domains. By applying this method to the experimental quenching titration data for the multi-domain binding of Cc to CcP, we were able to evaluate, for the first time, the functional relevance of the low-affinity domain in the binary and ternary complexes at both low and high ionic strength. At low ionic strength, essentially no Cc binds at the low-affinity domain in a binary complex. However, reactivity at this domain is significant and indeed can be dominant, in the ternary complex, which does form with measureable concentration. At higher, more physiologically-relevant ionic strengths, very little ternary complex forms, but now the situation is changed, and there is significant reactivity from the 1:1 complex with Cc bound at the low-affinity domain. In other words, there is substantial reactivity from Cc bound at the weakly binding domain over a wide range of ionic strengths, either in a binary or a ternary complex. Finally, we suggest that at high ionic strength, interactions between the two bound Cc are shielded such that it is easier to bind the second Cc molecule when one domain is occupied already.
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