Mechanical anisotropy is an important feature of materials. Depending on the direction it is pulled, a material can exhibit very different mechanical properties. Mechanical anisotropy on the microscopic scale has also been observed for individual elastomeric proteins. Depending upon the direction along which it is stretched, a protein can unfold via different mechanical unfolding pathways and exhibit vastly different mechanical stability. However, it remains to be demonstrated if the concept of mechanical anisotropy can be extended to the molecular scale for small molecular objects containing only a few chemical bonds. Here, we choose the iron-sulfur center FeS4 in the simplest iron-sulfur protein rubredoxin as a model system to demonstrate the molecular level mechanical anisotropy. We used single molecule atomic force spectroscopy to investigate the mechanical rupture of the FeS4 center along different pulling directions. The FeS4 cluster is a simple molecular object with defined three-dimensional structure, where a ferric ion and four coordinating cysteinyl ligands are arranged into a distorted tetrahedral geometry. Mutating two specific residues in rubredoxin to cysteines provides anchoring points that enable us to stretch and rupture the FeS4 center along five distinct and precisely controlled directions. Our results showed that the mechanical stability as well as the rupture mechanism and kinetics of the FeS4 center are strongly dependent upon the direction along which it is stretched, suggesting that the very small and simple FeS4 center exhibits considerable mechanical anisotropy. It is likely that structural asymmetry in the FeS4 cluster and the modulation of the local environment due to partial unfolding of rubredoxin are responsible for the observed mechanical anisotropy. Our results suggest that mechanical anisotropy is a universal feature for any asymmetrical three-dimensional structure, even down to a molecular scale, and such mechanical anisotropy can be potentially utilized to control the mechanochemical reactivity of molecular objects.
