Shedding Light on the Electron Delocalization Pathway at the [Fe2S2]2+ Cluster of FDX2

: In this paper, we investigate the electronic structure of the [Fe2S2]2+ cluster of human ferredoxin 2 by designing NMR experiments tailored to observe hyperfine-shifted and fast relaxing resonances in the immediate proximity of the cluster and adding a quantitative layer of interpretation through quantum chemical calculations. The combination of paramagnetic NMR and density functional theory data provides evidence of the way unpaired electron density map is at the origin of the inequivalence of the two iron(III) ferredoxin centers. An electron spin density transfer is observed between cluster inorganic sulfide ions and aliphatic carbon atoms, occurring via a C-H---S-Fe3+ interaction, suggesting that inorganic cluster sulfide ions have a significant role in the distribution of electron spin density around the prosthetic group. The extended assignment of 1H, 13C, and 15N nuclei allows the identification of all residues of the binding loop and provides an estimate of the magnetic exchange coupling constant between the two Fe3+ ions of the [Fe2S2]2+ cluster of 386 cm-1. The approach developed here can be extended to other iron-sulfur proteins, providing a crucial tool to uncover subtle differences in electronic structures that modulate the functions of this protein family.