Proton NMR spectra of the Rieske-type ferredoxin from Xanthobacter strain Py2 were recorded in both H2O and D2O buffered solutions at pH 7.2. Several well-resolved hyperfine-shifted 1H NMR signals were observed in the 90 to −20 ppm chemical shift range. Comparison of spectra recorded in H2O and D2O buffered solutions indicated that the signals at −11.4 (L) and −15.5 (M) ppm were solvent-exchangeable and thus were assigned to the two histidine Nε2H protons. The remaining observed signals were assigned based upon chemical shift, T1 values, and one-dimensional nuclear Overhauser effect (nOe) saturation transfer experiments to either CβH or CαH protons of cluster cysteinyl or histidine ligands. Upon oxidation of the [2Fe-2S] cluster, only two broad resonances were observed, indicating that the two Fe(III) ions are strongly antiferromagnetically coupled. In addition, the temperature dependence of each observed hyperfine-shifted signal in the reduced state was determined, providing information about the magnetic properties of the [2Fe-2S]1- cluster. Fits of the temperature data observed for each resonance to equations describing the hyperfine shift with their Boltzmann weighting factors provided a ΔEL value of 185 ± 26 cm-1 which, in turn, gives −2J as 124 cm-1. These data indicate that the two iron centers in the reduced [2Fe-2S] Rieske-type center are moderately antiferromagnetically coupled. The combination of these data with the available spectroscopic and crystallographic results for Rieske-type proteins has provided new insights into the role of the Rieske-type protein from Xanthobacter strain Py2 in alkene oxidation.