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.