Proton nuclear magnetic resonance investigation of the [2Fe-2S](1-)-containing ''Rieske-type'' protein from Xanthobacter strain Py2

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 H-1 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 solvents exchangeable and thus were assigned to the two histidine (NH)-H-epsilon 2 protons. The remaining observed signals were assigned based upon chemical shift, T-1 values, and one-dimensional nuclear Overhauser effect (nOe) saturation transfer experiments to either (CH)-H-beta or (CH)-H-alpha 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 Delta E-L 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.