The use of hybrid hemoglobin (Hb), with mesoheme substituted for protoheme, allows separate monitoring of the alpha or beta hemes along the allosteric pathway. Using resonance Raman (rR) spectroscopy in silica gel, which greatly slows protein motions, we have observed that the Fe-histidine stretching frequency, vFeHis, which is a monitor of heme reactivity, evolves between frequencies characteristic of the R and T states, for both alpha or beta chains, prior to the quaternary R-T and T-R shifts. Computation of vFeHis, using QM/MM and the conformational search program PELE, produced remarkable agreement with experiment. Analysis of the PELE structures showed that the vFeHis shifts resulted from heme distortion and, in the a chain, Fe-His bond tilting. These results support the tertiary two-state model of ligand binding (Henry et al., Biophys. Chein. 2002, 98, 149). Experimentally, the vFeHis evolution is faster for beta than for alpha chains, and pump-probe rR spectroscopy in solution reveals an inflection in the vFeHis time course at 3 mu s for beta but not for alpha hemes, an interval previously shown to be the first step in the R-T transition. In the a chain vFeHis dropped sharply at 20 ps, the final step in the R-T transition. The time courses are fully consistent with recent computational mapping of the R-T transition via conjugate peak refinement by Karplus and co-workers (Fischer et al., Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 5608). The effector molecule IHP was found to lower vFeHis selectively for a chains within the R state, and a binding site in the alpha 1 alpha 2 cleft is suggested.