Single‐Molecule Force Spectroscopy Reveals Stability of mitoNEET and its [2Fe2Se] Cluster in Weakly Acidic and Basic Solutions

The outer mitochondrial membrane protein mitoNEET (mNT) is a recently identified iron‐sulfur protein containing a unique Fe 2 S 2 (His) 1 (Cys) 3 metal cluster with a single Fe−N(His87) coordinating bond. This labile Fe−N bond led to multiple unfolding/rupture pathways of mNT and its cluster by atomic force microscopy‐based single‐molecule force spectroscopy (AFM‐SMFS), one of most common tools for characterizing the molecular mechanics. Although previous ensemble studies showed that this labile Fe−N(His) bond is essential for protein function, they also indicated that the protein and its [2Fe2S] cluster are stable under acidic conditions. Thus, we applied AFM‐SMFS to measure the stability of mNT and its cluster at pH values of 6, 7, and 8. Indeed, all previous multiple unfolding pathways of mNT were still observed. Moreover, single‐molecule measurements revealed that the stabilities of the protein and the [2Fe2S] cluster are consistent at these pH values with only ≈20 pN force differences. Thus, we found that the behavior of the protein is consistent in both weakly acidic and basic solutions despite a labile Fe−N bond.