Single molecule force spectroscopy reveals that iron is released from the active site of rubredoxin by a stochastic mechanism.

Metal centers in metalloproteins involve multiple metal-ligand bonds. The release of metal ions from metalloproteins can have significant biological consequences, so understanding of the mechanisms by which metal ion dissociates has broad implications. By definition, the release of metal ions from metalloproteins involves the disruption of multiple metal-ligand bonds, and this process is often accompanied by unfolding of the protein. Detailed pathways for metal ion release from metalloproteins have been difficult to elucidate by classical ensemble techniques. Here, we combine single molecule force spectroscopy and protein engineering techniques to investigate the mechanical dissociation mechanism of iron from the active site of the simplest iron-sulfur protein, rubredoxin, at the single molecule level. Our results reveal that the mechanical rupture of this simplest iron center is stochastic and follows multiple, complex pathways that include concurrent rupture of multiple ferric-thiolate bonds as well as sequential rupture of ferric-thiolate bonds that lead to the formation of intermediate species. Our results uncover the surprising complexity of the rupture process of the seemingly simple iron center in rubredoxin and provide the first unambiguous experimental evidence concerning the detailed mechanism of mechanical disruption of a metal center in its native protein environment in aqueous solution. This study opens up a new avenue to investigating the rupture mechanism of metal centers in metalloproteins with unprecedented resolution by using single molecule force spectroscopy techniques.