We present the results of Mossbauer and magnetization studies of [2Fe-2S](+) clusters from the wild-type and the C56S variant 2Fe-ferredoxin from Clostridium pasteurianum. At pH = 11 in Tris/Caps buffer, samples of the C56S variant contain, at 4.2 K, a 1:1 mixture of [2Fe-2S](+) clusters with valence localized S = 1/2 (Fd(1/2)) and valence-delocalized S = 9/2 (Fd(9/2)) ground states. A spin Hamiltonian analysis of Mossbauer spectra recorded in applied fields up to 8.0 T provides the fine structure and hyperfine parameters for Fd(9/2): D-9/2 = -1.5 cm(-1), E/D = 0.11, A = (-13, -13, -9.1) MHz, Delta E-Q = 1.83 mm/s, eta = 0, and delta = 0.50 mm/s. A comparative analysis of the hyperfine tensor components for Fd(9/2) and Fd(1/2) shows that the intrinsic A-values cannot be directly transferred from localized to delocalized systems. This result indicates that valence delocalization is accompanied by additional modifications in the electronic structure. High temperature Mossbauer studies show an increase in the fraction of valence-delocalized clusters, from ca. 50% at 4.2 K to ca. 94% at 200 K. Magnetic susceptibility studies rule out that the delocalized fraction generated at high temperature results from a spin conversion of Fd(1/2) to Fd(9/2) Rather, the change in the delocalized fraction is due to a rapid increase in the intramolecular electron-transfer rate between the two iron sites of Fd(1/2). Such a localization-to-delocalization transition has not been observed previously for any Fe-S cluster. The spectral features and the temperature range of the transition (approximate to 100 K) suggest a distribution in the rate of electron transfer between the iron sites of Fd(1/2) and point toward a dispersion in the values for the electronic parameters arising from the interaction of the cluster with the protein in different conformations. The rate for electron transfer between the iron sites of Fd(1/2) was calculated using a quantum mechanical method which takes into account Heisenberg-Dirac-van Vleck exchange, spin-dependent resonance interaction, and vibronic trapping. By assuming a distribution in the parameters characterizing these interactions, we have been able to model the temperature dependence of the fraction of delocalized Fd(1/2) molecules. Our studies suggest that electron-transfer rates in Fe-S clusters from ferredoxins may probe the conformational substates of the protein moiety.