Frataxin, mitochondrial
[2Fe-2S] cluster assembly
Mechanism and structural dynamics of sulfur transfer during de novo [2Fe-2S] cluster assembly on ISCU2.
Maturation of iron-sulfur proteins in eukaryotes is initiated in mitochondria by the core iron-sulfur cluster assembly (ISC) complex, consisting of the cysteine desulfurase sub-complex NFS1-ISD11-ACP1, the scaffold protein ISCU2, the electron donor ferredoxin FDX2, and frataxin, a protein dysfunctional in Friedreich's ataxia. The core ISC complex synthesizes [2Fe-2S] clusters de novo from Fe and a persulfide (SSH) bound at conserved cluster assembly site residues. Here, we elucidate the poorly understood Fe-dependent mechanism of persulfide transfer from cysteine desulfurase NFS1 to ISCU2. High-resolution cryo-EM structures obtained from anaerobically prepared samples provide snapshots that both visualize different stages of persulfide transfer from Cys381<sup>NFS1</sup> to Cys138<sup>ISCU2</sup> and clarify the molecular role of frataxin in optimally positioning assembly site residues for fast sulfur transfer. Biochemical analyses assign ISCU2 residues essential for sulfur transfer, and reveal that Cys138<sup>ISCU2</sup> rapidly receives the persulfide without a detectable intermediate. Mössbauer spectroscopy assessing the Fe coordination of various sulfur transfer intermediates shows a dynamic equilibrium between pre- and post-sulfur-transfer states shifted by frataxin. Collectively, our study defines crucial mechanistic stages of physiological [2Fe-2S] cluster assembly and clarifies frataxin's molecular role in this fundamental process.