Thermodynamic communication between protein substructures has been investigated by determining the stabilizing effect of mutations at position 52 in the least stable, N-yellow, substructure of cytochrome c on the second least stable, Red, and most stable, Blue, substructures of the protein. A Lys 73 → His (H73) variant of iso-1-cytochrome c, containing these mutations was used to measure the stability of the Red substructure of cytochrome c through the pH and guanidine hydrochloride (gdnHCl) dependence of the His 73-mediated alkaline conformational transition. The stability of the Blue substructure was measured by global unfolding with gdnHCl and increased by 1 to 3.5 kcal/mol versus the H73 variant. The data demonstrate that the increase in stability of the Red substructure is similar to the increase in global stability, consistent with upward propagation of stabilizing energy from less (N-yellow) to more stable (Red and Blue) protein substructures. The result also supports sequential rather than independent unfolding of the N-yellow and Red substructures of cytochrome c. The data indicate that a leucine at position 52 alters the nature of partial unfolding of the Red substructure, a surprising effect for a single-site mutation. For all variants, the thermodynamics of formation of the Lys 79 alkaline state, which does not unfold the entire Red substructure, shows less stabilization of the portion of the protein unfolded relative to the stabilization of the Blue substructure, indicating that propagation of energy between substructures is somewhat disrupted when unfolding does not correspond to a natural substructure.