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 57 Fe nuclei and protons of the high-potential iron−sulfur (HiPIP) protein iso-II from Ectothiorhodospira halophila in frozen solutions, i.e., on nonoriented systems, has been exploited to determine electronic and structural details of the oxidized [Fe 4 S 4 ] 3+ cluster and its protein environment. Two distinct 57 Fe hyperfine couplings were resolved and assigned to pairs of highly symmetric ferric and mixed-valence iron ions in agreement with results of Mössbauer and ENDOR studies on related proteins and model compounds. From the analysis of dipolar contributions of the eight cysteine β-CH 2 − 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 coefficients, which suggest the existence of a | 7 / 2,3, 1 / 2 〉 state or an admixture of | 9 / 2,4, 1 / 2 〉 and | 7 / 2,4, 1 / 2 〉 as possible ground states of the cluster. The identification of the mixed-valence and ferric irons within the cluster was in agreement with NMR results based on the sequence specific assignments of proton couplings. In addition, a unique orientation of the g -tensor with respect to the molecular frame was found in the protein, the maximal g -tensor component being nearly perpendicular to the cluster face containing the mixed-valence irons. The intermediate and minimal components were related to the vectors connecting the ferric and mixed-valence irons, respectively. The analysis of the isotropic parts of the cysteine β-CH 2 -proton interactions allowed establishment of a correlation with the NMR shifts of corresponding protons, obtained by applying different scaling factors for protons close to the ferric and mixed-valence pair, respectively. The empirical law used to describe the relationship between the geometric orientation of a CH bond and the observed isotropic interaction for these types of clusters could be verified.