Unpaired Electron Spin Density Distribution across Reduced [2Fe-2S] Cluster Ligands by ¹³C_β-Cysteine Labeling

Iron-sulfur clusters are one of the most versatile and ancient classes of redox mediators in biology. The roles that these metal centers take on are predominantly determined by the number and types of coordinating ligands (typically cysteine and histidine) that modify the electronic structure of the cluster. Here we map the spin density distribution onto the cysteine ligands for the three major classes of the protein-bound, reduced [2Fe-2S](His)_n(Cys)_4-n (n = 0, 1, 2) cluster by selective cysteine-¹³C_β isotope labeling. The spin distribution is highly asymmetric in all three systems and delocalizes further along the reduced Fe²⁺ ligands than the nonreducible Fe³⁺ ligands for all clusters studied. The preferential spin transfer onto the chemically reactive Fe²⁺ ligands is consistent with the structural concept that the orientation of the cluster in proteins is not arbitrarily decided, but rather is optimized such that it is likely to facilitate better electronic coupling with redox partners. The resolution of all cysteine-¹³C_β hyperfine couplings and their assignments provides a measure of the relative covalencies of the metal-thiolate bonds not readily available to other techniques.