The linked equilibria of an allosterically regulated protein are defined by the structures, residue-specific dynamics and global energetics of interconversion among all relevant allosteric states. Here, we use isothermal titration calorimetry (ITC) to probe the global thermodynamics of allosteric negative regulation of the binding of the paradigm ArsR-family zinc sensing repressor Staphylococcus aureus CzrA to the czr DNA operator (CzrO) by Zn2+. Zn2+ binds to the two identical binding sites on the free CzrA homodimer in two discernible steps. A larger entropic driving force Delta(-T Delta S) of -4.7 kcal mol(-1) and a more negative Delta C-p characterize the binding of the first Zn2+ relative to the second. These features suggest a modest structural transition in forming the Zn-1 state followed by a quenching of the internal dynamics on filling the second zinc site, which collectively drive homotropic negative cooperativity of Zn2+ binding (Delta(Delta G) = 1.8 kcal mol(-1)). Negative homotropic cooperativity also characterizes Zn2+ binding to the CzrA center dot CzrO complex (Delta(Delta G) = 1.3 kcal mol(-1)), although the underlying energetics are vastly different, with homotropic Delta(Delta H) and Delta(-T Delta S) values both small and slightly positive. In short, Zn2+ binding to the complex fails to induce a large structural or dynamical change in the CzrA bound to the operator. The strong heterotropic negative linkage in this system (Delta G(c)(t) = 6.3 kcal mol(-1)) therefore derives from the vastly different structures of the apo-CzrA and CzrA center dot CzrO reference states (Delta H-c(t) = 9.4 kcal mol(-1)) in a way that is reinforced by a global rigidification of the allosterically inhibited Zn-2 state off the DNA (T Delta S-c(t) = -3.1 kcal mol(-1), i.e., Delta S-c(t) > 0). The implications of these findings for other metalloregulatory proteins are discussed.