Mechanical Unfolding Pathway of the High-Potential Iron–Sulfur Protein Revealed by Single-Molecule Atomic Force Microscopy: Toward a General Unfolding Mechanism for Iron–sulfur Proteins
2018
Li, Jiayu | Li, Hongbin
High-potential iron–sulfur proteins (HiPIPs) are an important class of metalloproteins with a [4Fe–4S] cluster coordinated by four cysteine residues. Distinct from other iron–sulfur proteins, the cluster in HiPIP has a high reduction potential, making it an essential electron carrier in bacterial photosynthesis. Here, we combined single-molecule atomic force microscopy and protein engineering techniques to investigate the mechanical unfolding mechanism of HiPIP from Chromatium tepidum (cHiPIP). We found that cHiPIP unfolds in a two-step fashion with the protein sequence sequestered by the iron–sulfur center as a stable unfolding intermediate state. The rupture of the iron–sulfur center of cHiPIP proceeds in two distinct parallel pathways; one pathway involves the concurrent rupture of multiple iron–thiolate bonds, and the other one involves the sequential rupture of the iron–thiolate bonds. This mechanistic information was further confirmed by mutational studies. We found that the rupture of the iron–thiolate bonds in reduced and oxidized cHiPIP occurred in the range of 150–180 pN at a pulling speed of 400 nm/s, similar to that measured for iron–thiolate bonds in rubredoxin and ferredoxin. Our results may have important implications for understanding the general unfolding mechanism governing iron–sulfur proteins, as well as the mechanism governing the mechanical rupture of the iron–sulfur center.
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