STERN-VOLMER IN REVERSE - 2 1 STOICHIOMETRY OF THE CYTOCHROME-C CYTOCHROME-C PEROXIDASE ELECTRON-TRANSFER COMPLEX/

A reverse protocol for measurements of molecular binding and reactivit y by excited-state quenching has been developed in which the quencher, held at a fixed concentration, is titrated by a photoexcitable probe molecule whose decay is monitored. The binding stoichiometries, affini ties, and reactivities of the electron-transfer complexes between cyto chrome c (Cc) and cytochrome c peroxidase (CcP) were determined over a wide range of ionic strengths (4.5 to 118 millimolar) by the study of photoinduced electron-transfer quenching of the triplet excited state of zinc-substituted Cc (ZnCc) by Fe(3+)CcP. The 2:1 stoichiometry see n for the binding of Cc to CcP at low ionic strength persists at the p hysiologically relevant ionic strengths and likely has functional sign ificance. Analysis of the stoichiometric binding and rate constants co nfirms that one surface domain of CcP binds Cc with a high affinity bu t with poor electron-transfer quenching of triplet-state ZnCc, whereas a second binds weakly but with a high rate of electron-transfer quenc hing.