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

Abstract

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)(Cys) (n = 0, 1, 2) cluster by selective cysteine-C isotope labeling. The spin distribution is highly asymmetric in all three systems and delocalizes further along the reduced Fe ligands than the nonreducible Fe ligands for all clusters studied. The preferential spin transfer onto the chemically reactive Fe 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-C hyperfine couplings and their assignments provides a measure of the relative covalencies of the metal-thiolate bonds not readily available to other techniques.

HYSCORE Insights into the Distribution of the Unpaired Spin Density in an Engineered CuA Site in Azurin and Its His120Gly Variant

“…One can propose that copper couplings determined from the EPR spectra can be further supported by the hyperfine tensors of the coordinated nitrogens and of the C β atoms of the cysteine ligands. Those can be obtained from 15 N ENDOR and 13 C HYSCORE experiments in a manner similar to that used in refs and , respectively, for other systems. High-field ENDOR- and ELDOR-detected NMR can also be useful for this purpose .…”

EPR-Derived Structure of a Paramagnetic Intermediate Generated by Biotin Synthase BioB

“…Labeling at 13 C9 (Figure A) gives the largest HFI of A = [1.30, 0.85, 4.98] MHz, corresponding to a iso = 2.37 MHz and T = 1.31 MHz, which is comparable to the HFI ( A = [1.8, 2.0, 5.1] MHz) of the carbon atom (C3) coordinated to the [4Fe–4S] cluster in the reaction intermediate of IspG . The a iso of 13 C9 is larger than the values of β- 13 C of cysteines coordinated to the ferric site of other reduced [2Fe–2S] + clusters (see Table ). We also determined the absolute sign of 13 C9 hyperfine coupling via VMT-ENDOR experiment; as shown in Figure D, the positive HFI suggests it originates from through-bond spin delocalization .…”

“…reaction intermediate of IspG. 63 The a iso of 13 C9 is larger than the values of β-13 C of cysteines coordinated to the ferric site of other reduced [2Fe−2S] + clusters 64 (see Table 5). We also determined the absolute sign of 13 C9 hyperfine coupling via VMT-ENDOR experiment; as shown in Figure 5D, the positive HFI suggests it originates from through-bond spin delocalization.…”

ATP Binding and a Second Reduction Enables a Conformationally Gated Uphill Electron Transfer

“…The determined e – max of 0.5 with ATP indicates comparable redox potentials of the cobalt and the [2Fe2S] cluster in the protein complex Co­(I)­FeSP:RACo ox :ATP. Various mechanisms can modulate the redox potential of the acceptor and/or donor cofactor, such as the change of the angles or bond length of the corrin ring, the position of the electron in the [2Fe2S] cluster, or H bond interactions of the two redox centers. , It seems likely that ATP and ADP binding changes one or several of these factors to a different extent thereby triggering electron transfer but resulting in different Δ E values for the cofactors and therefore different electron-transfer equilibria.…”