Mechanical Unfolding of the High Potential Iron-Sulfur Protein Probed by Single Molecule Atomic Force Microscopy

Abstract

High potential iron-sulfur protein (HiPIP) is an important category of metalloprotein with a 4Fe-4S cluster center coordinated by four cysteines. Different 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 used single molecule force spectroscopy to investigate the mechanical unfolding of HiPIP and the rupture of the ferric-thiolate bond under different redox potential. We found that the unfolding of HiPIP could be triggered by the rupture of the ferric-thiolate bonds at forces of ∼150 pN, which was lower than those for other iron-sulfur proteins, such as rubredoxin and ferredoxin. Both one-step and two-step unfolding processes were observed in our experiments, indicative of multiple mechanical unfolding pathways for HiPIP. In addition, the oxidation states of the metal center have little effect on the unfolding behavior of HiPiP. Further mutational studies will allow us to elucidate the complete rupture mechanism of the iron-sulfur center and the mechanical unfolding pathways of HiPIP.