The static structure of hemoglobin and its functional properties are very well characterized. It is still not known how energy is stored and used within the structure of the protein to promote function and functional diversity. An essential part of this question is understanding the mechanism through which the overall protein structure (quaternary structure) couples to the local environment about the oxygen binding sites. Time-resolved resonance Raman spectroscopy has been used to probe the vibrational degrees of the freedom of the binding site as a function of protein structure. Comparison of the spectra from both equilibrium and transient forms of deoxy hemoglobin from a variety of mammalian, reptilian, and fish hemoglobins reveals that for each quaternary structure there exist two tertiary states stabilized by the presence or absence of an iron-bound ligand. Pulse-probe Raman experiments show that for photodissociated, ligated hemoglobins the local tertiary structure relaxes at a solution-dependent rate extending from tens of nanoseconds to microseconds. In this local environment, the linkage between the iron and the proximal histidine proves to be the single observed structural feature that responds in a systematic and substantial manner to structural changes in the protein. The additional finding of a correlation between the frequency of the iron-proximal histidine stretching motion (nu Fe-His) and various parameters of ligand reactivity, including geminate recombination, implicates the associated localized structural element in the mechanism of protein control of ligand binding. On the basis of these and related finds, a model is presented to account for both coarse and fine control of ligand binding by the protein structure.