CYTOCHROME-C PEROXIDASE BINDS 2 MOLECULES OF CYTOCHROME-C - EVIDENCE FOR A LOW-AFFINITY, ELECTRON-TRANSFER-ACTIVE SITE ON CYTOCHROME-C PEROXIDASE

We have studied the affinity and stoichiometry of binding of cytochrom e c (Cc) to zinc-substituted cytochrome c peroxidase [(ZnP)CcP], which is structurally and electrostatically equivalent to ferrous CcP. Tran sient absorption spectroscopy has been used to measure both the total quenching of the triplet-state (ZnP)CcP [3(ZnP)CcP] by Fe3+Cc and the fraction of that quenching that is due to electron transfer (et). This redox quenching results in the formation of an intermediate (I) conta ining the zinc porphyrin pi-cation radical [(ZnP)+CcP] and Fe2+Cc. In titrations of (ZnP)CcP with Fe3+Cc(F) at low ionic strength, where F r epresents the fungal cytochromes c from Candida krusei, Pichia membran efaciens, or the yeast protein iso-1, the appearance of the et interme diate lags behind the total quenching, with appreciable formation of I occurring only for Cc to CcP ratios > 1. This behavior results from t he formation of a 2:1 complex, where one Fe3+Cc(F) binds to a high-aff inity domain that exhibits strong quenching yet is et-inactive, while the second Fe3+Cc(F) binds to a low-affinity domain that allows effici ent et quenching. At constant concentrations of both proteins, raising the ionic strength eliminates most of the et quenching but reduces th e total quenching only minimally, confirming that et occurs preferenti ally at the low-affinity binding domain, which is the more sensitive t o ionic strength. Analogous experiments also favor a 2:1 binding stoic hiometry for horse Cc[Cc(horse)] at low ionic strength, with et quench ing again proceeding much more favorably in the 2:1 complex than in th e 1:1 complex, as with Cc(F). However, the Fe3+Cc(horse) quenches only by electron transfer, unlike the Cc(F). The decay of the triplet-stat e (ZnP)CcP or magnesium-substituted CcP [(MgP)CcP] was examined during titrations with Fe3+Cc to determine limits for the dissociation rate constant (k(off)) for the complex. Fe3+Cc(horse) bound to the high-aff inity domain in a 1:1 complex at low ionic strength is in rapid exchan ge, with k(off) > 50 s-1, whereas Fe3+Cc(F) has k(off) < 200 s-1. Both types of Fe3+Cc have k(off) >10(4) s-1 when they are bound to the low -affinity domain in a 2:1 complex, at both low and high ionic strength s. In contrast, when in the ferrous form, both types of Cc have much l ower values of k(off) (<10 s-1) at low ionic strength when bound to th e low-affinity domain. The low limit of k(off) (<200 s-1) for Fe3+Cc(F ) at low ionic strength indicates that a simple one-site mechanism can not account for the much higher values found for the turnover number f or Cc(F:iso-1) [Erman, J. E., Kang, D. S., Kim, K. L., Summers, F. E., Matthis, A. L., & Vitello, L. B. (1991) Mol. Cryst. Liq. Cryst. 194, 253-258]. Confirming the proposal of Kang et al. [Kang, C. H., Ferguso n-Miller, S., & Margoliash, E. (1977) J. Biol. Chem. 252, 919-926], a model of CcP function which accounts for all of these results includes two distinct binding domains for Cc: a poorly reactive high-affinity domain with multiple, overlapping sites for Cc and a highly reactive l ow-affinity domain elsewhere on the peroxidase.