Present in most organisms, hexacoordinate hemoglobins (hxHbs) are proteins that have evolved the capacity for reversible bis-histidyl heme coordination. The heme prosthetic group of heme proteins enables diverse protein functionality, such as electron transfer, redox reactions, ligand transport, and enzymatic catalysis. The reactivity of heme is greatly affected by its coordination and the non-covalent chemical environment imposed by its connate protein and also by the nature of ligands present at the protein active center. Of considerable interest is how the hxHb globin fold achieves reversible intramolecular coordination while still enabling high-affinity binding of oxygen, nitric oxide, and other small ligands. Here this property is explored by examining the role of the protein matrix on coordination behavior in a group of hxHbs from animals, plants, and bacteria. This is done with a set of experiments measuring the reduction potentials of each wild type hxHb; its corresponding mutant protein where the reversibly bound histidine (the distal His) has been replaced with a non-coordinating side chain; and the mutant proteins saturated with exogenous imidazole, enable to assess the effects of the protein matrices on histidine coordination. The results show that the globin moiety of hxHb demonstrates flexible regulation of hexacoordination. The effect of ligand binding to the ferrous form of hxHbs is also studied by measuring their redox potentials in presence and absence of CO. The outcome of this study could be utilized to evaluate their potential physiological function in the context of ligand binding.