The cleavage of [4Fe-4S]-type clusters is thought to be important in proteins such as Fe-S scaffold proteins and nitrogenase. However, most [4Fe-4S]21 Clusters in proteins have two anti ferromagnetic ally coupled high-spin layers in which a minority spin is delocalized in each layer, thus forming a symmetric Fe2.5+-Fe2.5+ pair, and how cleavage occurs between the irons is puzzling because of the shared electron. Previously, we proposed a novel mechanism for the fission of a [4Fe-4S] core into two [2Fe-2S] cores in which the minority spin localizes on one iron, thus breaking the symmetry and creating a transition state with two Fe3+-Fe2+ pairs. Cleavage first through the weak Fe2+-S bonds lowers the activation energy. Here, we propose a test of this mechanism: break the symmetry of the cluster by changing the ligands to promote spin localization, which should enhance reactivity. The cleavage reactions for the homoligand [Fe4S4L4](2-) (L = SCH3, Cl, H) and heteroligand [Fe4S4(SCH3)(2)L-2](2-) (L = Cl, H) clusters in the gas phase were examined via broken-symmetry density functional theory calculations. In the heteroligand clusters, the minority spin localized on the iron coordinated by the weaker electron-donor ligand, and the reaction energy and activation barrier of the cleavage were lowered, which is in accord with our proposed mechanism and consistent with photoelectron spectroscopy and collision-induced dissociation experiments. These studies suggest that proteins requiring facile fission of their [4Fe-4S] cluster in their biological function might have spin-localized [4Fe-4S] clusters.