EVIDENCE FOR DAMPED HEMOGLOBIN DYNAMICS IN A ROOM-TEMPERATURE TREHALOSE GLASS

Upon photodissociation of its ligand, COHbA exhibits a wide range of n onequilibrium relaxation phenomena that start within a fraction of a p icosecond and extend out to tens of microseconds. In addition, equilib rium fluctuations of the protein result in conformational averaging. A ll of these dynamics can have an impact on Ligand rebinding, In all ef fort to better understand the relationship between conformational dyna mics and ligand-binding reactivity, COHbA was embedded in a room tempe rature trehalose sugar glass (Hagen et al. Science 1995, 269, 959) in order to uncouple solvent motions from protein dynamics as well as red uce the amplitude of large-scale protein conformational fluctuations. Time-resolved resonance Raman spectroscopy and ligand-rebinding kineti cs show that the trehalose glass does not impede the initial fast rela xation of the iron-histidine linkage, bur does dramatically impede con formational averaging and completely eliminates ligand escape at all t emperatures from 140 K to room temperature. Fluorescence measurements indicate that in the trehalose glass the picosecond tryptophan lifetim es are nearly unchanged, bur there is a complete absence of the nanose cond fluorescence decay (observed in aqueous solutions), which is repl aced by a decay of similar to 700 ps. This change in the fluorescence decay is ascribed to a significant decrease in the structural dynamics that normally allow transient opening of the distal heme pocket.