Probing structural and electronic properties of the oxidized [Fe4S4](3+) cluster of Ectothiorhodospira halophila iso-II high-potential iron-sulfur protein by ENDOR spectroscopy

The ENDOR response of Fe-57 nuclei and protons of the high-potential iron-s ulfur (HiPIP) protein iso-II from Ectothiorhodospira halophila in frozen so lutions, i.e., on nonoriented systems, has been exploited to determine elec tronic and structural details of the oxidized [Fe4S4](3+) cluster and its p rotein environment. Two distinct Fe-57 hyperfine couplings were resolved an d assigned to pairs of highly symmetric ferric and mixed-valence iron ions in agreement with results of Mossbauer and ENDOR studies on related protein s and model compounds. From the analysis of dipolar contributions of the ei ght cysteine beta-CH2- and five additional protons of residues close to the cluster, the spin population on the iron ions in the ferric and the mixed- valence pair was deduced. The symmetric spin vector coupling model yields c oefficients, which suggest the existence of a \7/2,3,1/2] State or an admix ture of \9/2,4,1/2] and \7/2,4,1/2] as possible ground states of the cluste r. The identification of the mixed-valence and ferric irons within the clus ter was in agreement with NMR results based on the sequence specific assign ments of proton couplings. In addition, a unique orientation of the g-tenso r with respect to the molecular frame was found in the protein, the maximal g-tensor component being nearly perpendicular to the cluster face containi ng the mixed-valence irons. The intermediate and minimal components were re lated to the vectors connecting the ferric and mixed-valence irons, respect ively. The analysis of the isotropic parts of the cysteine beta-CH2-proton interactions allowed establishment of a correlation with the NMR shifts of corresponding protons, obtained by applying different scaling factors for p rotons close to the ferric and mixed-valence pair, respectively. The empiri cal law used to describe the relationship between the geometric orientation of a CH bond and the observed isotropic interaction for these types of clu sters could be verified.