A hierarchy of functionally important relaxations within myoglobin based on solvent effects, mutations and kinetic model.

Geminate CO rebinding in myoglobin is studied for two viscous solvents, trehalose and sol-gel (bathed in 100% glycerol) at several temperatures. Mutations in key distal hemepocket residues are used to eliminate or enhance specific relaxation modes. The time-resolved data are analyzed with a modified Agmon-Hopfield model which is capable of providing excellent fits in cases where a single relaxation mode is dominant. Using this approach, we determine the relaxation rate constants of specific functionally important modes, obtaining also their Arrhenius activation energies. We find a hierarchy of distal pocket modes controlling the rebinding kinetics. The "heme access mode" (HAM) is responsible for the major slow-down in rebinding. It is a solvent-coupled cooperative mode which restricts ligand return from the xenon cavities. Bulky side-chains, like those His64 and Trp29 (in the L29W mutant), operate like overdamped pendulums which move over and block the binding site. They may be either unslaved (His64) or moderately slaved (Trp29) to the solvent. Small side-chain relaxations, most notably of leucines, are revealed in some mutants (V68L, V68A). They are conjectured to facilitate inter-cavity ligand motion. When all relaxations are arrested (H64L in trehalose), we observe pure inhomogeneous kinetics with no temperature dependence, suggesting that proximal relaxation is not a factor on the investigated timescale.