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Single-Molecule Force Spectroscopy Reveals that the Fe–N Bond Enables Multiple Rupture Pathways of the 2Fe2S Cluster in a MitoNEET Monomer

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posted on 20.10.2020, 19:16 by Guobin Song, Xuan Ding, Huaxing Liu, Guodong Yuan, Fang Tian, Shengchao Shi, Yang Yang, Guoqiang Li, Peng Zheng
The mitochondrial outer membrane protein, mitoNEET (mNT), is an iron–sulfur protein containing an Fe2S2(His)1(Cys)3 cluster with a unique single Fe–N bond. Previous studies have shown that this Fe­(III)–N­(His) bond is essential for metal cluster transfer and protein function. To further understand the effect of this unique Fe–N bond on the metal cluster and protein, we used atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS) to investigate the mechanical unfolding mechanism of an mNT monomer, focusing on the rupture pathway and kinetic stability of the cluster. We found that the Fe–N bond was the weakest point of the cluster, the rupture of which occurred first, and could be independent of the cluster break. Moreover, this Fe–N bond enabled a dynamic and labile iron–sulfur cluster, as multiple unfolding pathways of mNT with a unique Fe2S2(Cys)3 intermediate were observed accordingly.