Cytochrome c Peroxidase Binds Two Molecules of Cytochrome c: Evidence for a Low-Affinity, Electron-Transfer-Active Site on Cytochrome c Peroxidase

Eric D.A. Stemp, Brian M. Hoffman*

*Corresponding author for this work

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91 Scopus citations


We have studied the affinity and stoichiometry of binding of cytochrome c (Cc) to zinc-substituted cytochrome c peroxidase [(ZnP)CcP], which is structurally and electrostatically equivalent to ferrous CcP. Transient 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) containing the zinc porphyrin π-cation radical [(ZnP)+CcP] and Fe2+Cc. In titrations of (ZnP)CcP with Fe3+Cc(F) at low ionic strength, where F represents the fungal cytochromes c from Candida krusei, Pichia membranefaciens, or the yeast protein iso-1, the appearance of the et intermediate lags behind the total quenching, with appreciable formation of I occurring only for Cc to CcP ratios > 1. This behavior results from the formation of a 2:1 complex, where one Fe3+Cc(F) binds to a high-affinity domain that exhibits strong quenching yet is et-inactive, while the second Fe3+Cc(F) binds to a low-affinity domain that allows efficient et quenching. At constant concentrations of both proteins, raising the ionic strength eliminates most of the et quenching but reduces the total quenching only minimally, confirming that et occurs preferentially at the low-affinity binding domain, which is the more sensitive to ionic strength. Analogous experiments also favor a 2:1 binding stoichiometry for horse Cc [Cc(horse)] at low ionic strength, with et quenching again proceeding much more favorably in the 2:1 complex than in the 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-state (ZnP)CcP or magnesiumsubstituted CcP [(MgP)CcP] was examined during titrations with Fe3+Cc to determine limits for the dissociation rate constant (koff) for the complex. Fe3+Cc(horse) bound to the high-affinity domain in a 1:1 complex at low ionic strength is in rapid exchange, with koff > 50 s−1 whereas Fe3+Cc(F) has koff < 200 s1. Both types of Fe3+Cc have koff > 104s1 when they are bound to the low-affinity domain in a 2:1 complex, at both low and high ionic strengths. In contrast, when in the ferrous form, both types of Cc have much lower values of koff (<10 s−1) at low ionic strength when bound to the low-affinity domain. The low limit of koff (<200 s−1) for Fe3+Cc(F) at low ionic strength indicates that a simple one-site mechanism cannot account for the much higher values found for the turnover number for 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., Ferguson-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 low-affinity domain elsewhere on the peroxidase.

Original languageEnglish (US)
Pages (from-to)10848-10865
Number of pages18
Issue number40
StatePublished - 1993

ASJC Scopus subject areas

  • Biochemistry


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