Mechanism and structural dynamics of sulfur transfer during de novo [2Fe-2S] cluster assembly on ISCU2

Abstract

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 assem-bly site residues. Here, we elucidated 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 provided snapshots that both visualized differ-ent stages of persulfide transfer from Cys381NFS1 to Cys138ISCU2 and clarified the molecular role of frataxin in optimally positioning assembly site residues for sulfur transfer. Biochemical analyses assigned ISCU2 residues essential for sulfur transfer, and revealed that Cys138ISCU2 rapidly received the persulfide without a detectable intermediate. Mössbauer spectroscopy assessing the Fe coordination of various sulfur transfer intermediates showed a dynamic equilibrium between pre- and post-sulfur-transfer states shifted by frataxin. Collectively, our study defined crucial mechanistic stages of physiological [2Fe-2S] cluster assembly and clarified frataxin’s molecular role in this fundamental process.