Determination of the formal reduction potential, the electron stoichiometry, and the magnitude of the conformational change upon reduction for cytochrome c from potential-dependent fluorescence spectra.

A long-optical-path electrochemical cell was used to determine the formal reduction potential (E0') and the electron stoichiometry (n) for 1 microM horse heart cytochrome c solutions from Nernst plots of the fluorescence spectrum of the tryptophan-59. Various concentration ratios of the oxidized to the reduced forms of the protein were generated by application of different potentials. The fluorescence spectrum was found to decrease with increasing concentrations of reduced cytochrome c. This decrease is interpreted in terms of a movement of the tryptophan toward the heme upon reduction, resulting in an increase in the rate of energy transfer to the heme. The magnitude of the conformational change is compared for cytochrome c in NaCl-H2O and NaBr-H2O solutions with the latter being larger.