Electron Paramagnetic Resonance Insights into Direct Electron Transfer Between FDX1 and Elesclomol‐Cu²⁺ Complex in Cuproptosis

Copper-induced cell death, known as cuproptosis, is distinct from other forms of cell death processes. Previous studies have proposed Elesclomol (ES) as a potent cuproptosis inducer, with the mitochondrial ferredoxin protein FDX1 postulated as its primary target. However, the mechanistic details of electron transfer (ET) between Elesclomol-Cu²⁺ (ES-Cu²⁺) and FDX1 remain elusive, particularly lacking direct spectroscopic evidence. Here, electron paramagnetic resonance (EPR) spectroscopy was employed to characterize the direct ET process between FDX1 and ES-Cu²⁺ complexes. Comprehensive low-temperature EPR measurements validated the efficient ET from reduced FDX1 (FDX1_red) to oxidized ES-Cu²⁺ complex both in vitro and in vivo. Moreover, ES-Cu²⁺ complex exhibited a greater propensity for accepting electrons from FDX1 compared to free copper ions (Cu²⁺), demonstrating the high efficiency of ES-Cu²⁺ as a primary electron scavenger for FDX1 in cells. In addition, EPR power saturation experiments and molecular docking analysis indicated that FDX1 has a much higher binding affinity for ES-Cu²⁺ than its homologue FDX2, providing crucial insights into the functional specificity between FDX1 and FDX2. This work advances our understanding of ET processes in cuproptosis and underscores the significant potential of EPR spectroscopy in the study of important redox events in cells.