Directional information flow in human frataxin defines allosteric pathways connecting the hydrophobic core to the iron‐binding ridge

Frataxin is a mitochondrial iron‐binding protein whose deficiency causes Friedreich's ataxia, yet the dynamic mechanisms by which this protein communicates iron‐binding events to distal regions remain poorly understood. Traditional correlation‐based analyses identify coupled residue motions but cannot resolve the directionality of information flow, leaving critical mechanistic gaps. Here we employ transfer entropy analysis of molecular dynamics simulations (1.5 μs) to map the complete allosteric network in human frataxin, validated through orthogonal experimental approaches. We identify LEU47 (LEU136 in UniProt Q16595 numbering) and LEU51 (LEU140) as primary signal sources with net transfer entropy values of 0.415 and 0.249, respectively, connecting the hydrophobic core to the iron‐binding acidic ridge. NMR relaxation at 600 and 800 MHz reveals elevated R ₂ /R ₁ ratios (9.90–10.00) and significant exchange contributions (Rex = 3–5 s ⁻¹ ) specifically at these primary signal source residues, indicating μs–ms dynamics. Hydrogen–deuterium exchange mass spectrometry demonstrates that hub residues possess intermediate protection factors (ln(PF) = 5.97–6.07) optimal for conformational signaling, while iron binding induces bidirectional protection changes propagating through the identified pathway. Systematic mutagenesis confirms that disruption of hub residues reduces iron‐binding affinity 1.9–4.2‐fold and decreases thermal stability by 4.3–11.2 °C, despite occupying buried‐core positions distant from the iron‐coordinating acidic‐ridge residues (LEU136/LEU140 Cα to ASP122, ASP124, and GLU189 = 6.7 to 11.8 Å in PDB 1EKG ). The strong prediction–experiment correlation establishes transfer entropy as a reliable predictor of functionally important allosteric residues and provides a methodological framework applicable to other proteins of biomedical significance.