Cytochrome c peroxidase (CcP) binds cytochrome c (Cc) at two distinct surface binding domains, one having high affinity for Cc and the other having low affinity. The identity of the surface binding domains on CcP has been probed by studying photoinduced interprotein heme-heme electron transfer (ET) between Zn-substituted horse Cc, ZnCc, and a cloned cytochrome c peroxidase, CcP(D37K). Charge-reversal substitution of the negatively-charged residue Asp 37 of CcP by a positively-charged residue lysine greatly decreases the stoichiometric constant for 1:1 binding of Cc, from 8.5 x 10(5) M(-1) for the wild-type CcP to 1.2 x 10(4) M(-1) for CcP(D37K) (mu = 18 mM), thereby identifying residue 37 as part of the high-affinity binding domain of CcP(WT) (domain 1). This assignment is consistent with domain 1 as being that observed in a crystal of the Cc-CcP complex. The diminished ability of CcP(D37K) to bind Cc at domain 1 also suppresses binding of a:second Cc molecule to form a ternary complex. However, the mutation sharply increases the reactivity of CcP : the stoichiometric rate constant for heme-heme ET within the 1:1 (3)ZnCc-Fe(3+)CcP(D37K) complex is 4000 s(-1), much higher than that (40 s(-1)) for ZnCc(II)-Fe3+-CcP(WT). These results are explained by two-domain binding, where the high-affinity domain 1 has low heme-heme ET reactivity, while domain 2 has high heme-heme ET reactivity. The CcP(D37K) mutant exhibits greater reactivity than CcP(WT) because the mutational weakening of Cc-binding at CcP domain 1 increases the fraction of 1:1 complex where Cc is bound at the heme-reactive domain 2. Upon changing the ionic strength from 18 to 59 mN, the stoichiometric ET rate constant increases for the Cc-CcP(WT) complex, whereas it decreases 4-fold for the Cc(H)-CcP(D37K) complex, which indicates that the two distinct binding domains on CcP are differentially affected by ionic strength. This overall picture of ET between these two proteins is supported by a careful reconsideration of the most recent work from other laboratories. We hypothesize that domain 1 may make the dominant contribution to the direct reduction of the Trp 191 radical, while domain 2 provides the dominant kinetic site for ferryl-heme reduction.