The functional importance of large-scale motions and transitions of carbonmonoxy myoglobin (MbCO) conformational substates (Cs) has been studied by molecular dynamics (MD) and conformational flooding (CF) simulations. A flooding potential was constructed from an 800 ps MD trajectory of solvated MbCO to accelerate slower protein motions beyond the time scale of contemporary simulations. Two conformational transitions (tier-l substates) resulting from seven principal molecular motions were assigned to the spectroscopic Ao state (tier-0 substate) of MbCO, where His64 is solvated and nor within the hydrophobic pecker binding site. The first computed conformational transition involves a distal pocket gate defined by the C and D helices and the interconnecting CD loop (residues 40-55). The gate-like motion is interpreted to regulate ligand access from the distal side of the hems. Simultaneously, a proximal pocket lever involving the F helix and surrounding EF and FG loops (residues 82-105) is found to shuttle the heme deep into the protein matrix (heme rmsd of 3.9 Angstrom) as the distal pocket gate opened. The lever's effect on the heme motion is assumed to attract ligands into the heme pocket. The second major transition involves the compression and expansion of the cavity formed by the EF loop (residues 77-84) and the GH loop and H helix (residues 122-138). The motion is interpreted to modulate the hydrophobic pocket volume and regulate the ligand access from the proximal side of the heme. A third computed conformational transition was Found to be a combination of the previous motions. For the first time, CF was applied in a series of room temperature simulations to accelerate molecular motions of the MbCO native fold and define the lower tier hierarchy of substate structure. The computed CSs and associated transitions coincide with previously suggested putative ligand escape pathways, and support a hierarchical description of protein dynamics and structure. A unified model that utilizes both mechanisms of distal His64 modulation (tier-0) and protein equilibrium fluctuations (tier-l) is presented to explain ligand diffusion in the MbCO dissociation reaction.