| Summary: | The temperature, T, (140 K to 320 K) and pressure, P, (1 00 kPa to 100 MPa) dependence of the CO stretching bands in Sperm Whale Myoglobin, Mb, in various solvents has been studied with FTIR spectroscopy. The data are consistent with hierarchically ordered conformational substates, csn' (nth tier), for the protein-ligand-solvent system. The spectra can be resolved into three bands: Ao at -1966 cm-1, A1 at -1946 cm-1 and A3 at -1933 cm-1. The areas of the A bands are correlated with cso. Fits to the relative areas of the A bands as a function of T and P yield the relative energies, entropies and volumes of the A bands, cso. The width, n, and peak frequency, Vi, of each Ai is correlated with a sub-subset of CSs, CS1. Below about 180K in 75°/o glycerol-water, the system is frozen into a glassy state. Calorimetry measurements using a differential scanning calorimeter, DSC, give further evidence of the glass transition, GT. The dynamic responce of the A bands following a P-release (1 OOMPa to 7MPa), was also studied from -1 Os to -1 04s in the GT range of 170 K to 210 K. Between 170 K and 195 K, ro narrows and vo shifts towards their low P values, indicating internal redistribution of Ao, (functionally important motion within tier 1, fim1 }. lnterconversion of A1 with A3 is also measured in this T range, fim0A1 <->A3· Above -195 K, the A substates show rapid internal redistribution and A1 and A3 are in equilibrium, both within 1 Os. Between 195 K and 210 K, slow interconversion of Ao with A1 and A3 is observed, fim0AO<->A1 ,A3. The relaxations show similarities with the dynamics of glasses and spin glasses: (i) the observed relaxations are non-exponential in time indicating a distribution of relaxation rates. Each A substate thus can consist of many sub-substates, CS1; the relaxations "freeze" out within a narrow T range of -25 K with Arrhenius rates having barrier heights between 125 and 135 kJ mol-1 and prefactors of -1040s-1, indicating a cooperative many-bodied phenomenon.
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