Multi-domain binding of cytochrome c peroxidase by cytochrome c: Thermodynamic vs. microscopic binding constants

We have demonstrated that cytochrome c (Cc) binds to cytochrome c peroxidas e (CcP) with a 2:1 stoichiometry, and that: (i) Cc reacts at two distinct a nd 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, howev er, provide only stoichiometric (thermodynamic) parameters. They do not dir ectly probe the microscopic properties of the individual domains, and thus do not apportion the reactivity due to 1:1 stoichiometry between the two ty pes 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 a nalyzing the stoichiometric parameters obtained from triplet quenching titr ations to partition the quenching of ZnCc by CcP into contributions from th e binary and ternary complexes, and also to give limits on the partition of the quenching into contributions from the low- and high-affinity binding d omains. By applying this method to the experimental quenching titration dat a 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 io nic strength, essentially no Cc binds at the low-affinity domain in a binar y complex. However; reactivity at this domain is significant and indeed can be dominant, in the ternary complex, which does form with measureable conc entration. At higher, more physiologically relevant ionic strengths, very l ittle 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-affin ity domain. In other words, there is substantial reactivity from Cc bound a t 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 stre ngth, interactions between the two bound Cc are shielded such that it is ea sier to bind the second Cc molecule when one domain is occupied already.