Cyclic voltammetry (CV), W-visible (W-vis), circular dichroism (CD), and resonance Raman (lambda(ex) = 406.7, 413.1 nm) spectroscopy have been used to probe the structure and redox function of horse cytochrome c (cyt c) in aqueous mixtures of three water-miscible organic solvents, specifically, acetonitrile (ACN), dimethylformamide (DMF), and dimethyl sulfoxide (DMSO) containing 100-60% water. As the concentration of the organic solvent is increased, significant changes are observed in the spectroscopy of ferricytochrome c (ferricyt c) with the greatest structural changes observed for ferricyt c in mixed solvent media with the lowest dielectric constant (30% ACN in this work). In the UV-visible spectrum, the Soret band blue shifts (1 nm) and the intensity of the 695 nm band decreases. UV CD (185-240 nm) suggest that changes in the protein secondary structure on going from aqueous to nonaqueous media are relatively small and that the protein structure remains largely intact in nonaqueous media. In the visible CD spectrum, the negative 417 nm CD signal disappears, signaling significant changes in heme-polypeptide interactions. Shifts in the vibrational frequencies and changes in the relative intensities of bands in both the marker band and low frequency spectral regions of cyt c in mixed media have been attributed to mixtures of the type IVa, IVb, Va, and Vb alkaline conformers of ferricyt c in mixed media. The change in the composition of these mixtures as the dielectric constant decreases parallels closely that reported by Dopner et al.(1) for yeast iso-1-cytochrome c in aqueous solution as the aqueous solution pH is raised from pH 7 to pH 10. The spectroscopic characteristics of the reduced form of cyt c in mixed solvents are very similar to those of native ferrous cyt c (ferrocyt c) in aqueous solution, reflecting minimal change in heme active site structure in the reduced state. Cyt c in mixed solvents exhibits a quasireversible, one-electron response at 4,4'-dipyridyl disulfide-modified Au electrodes between 5 and 200 mV/s. The redox potential for cyt c in mixed media (199-274 mV vs SHE) and the rate of heterogeneous electron transfer (0.4-3.5 x 10(-3) cm/s) decrease as the organic solvent content of the solvent medium increases. Our data support the conclusion that the internal dielectric constant within the heme crevice plays the kev role in determining the reduction potential of cyt c and suggest that the effects of axial ligation (Met vs Lys) may be less significant than previously believed.