Structural heterogeneity and ligand binding in carbonmonoxy myoglobin crystals at cryogenic temperatures

We have characterized the ligand-rebinding behavior of single crystal native sperm whale carbonmonoxy myoglobin (swMbCO) (space group P21) and a synthetic mutant swMbCO (space group P6) at cryogenic temperatures by using temperature-derivative spectroscopy (TDS) with monitoring of the CO stretch bands in the mid-infrared. Crystals were studied at pH 5.1 and 7.0 for native swMbCO and at pH 7.0 for the mutant; both short-flash and extended illumination protocols were performed. The TDS analysis yields the enthalpy barrier distributions for recombination in the individual taxonomic (A) substates, A0, A1, and A3. A single gaussian barrier distribution gave a good first-order description but was insufficient to precisely fit the data within each substate. An additional minority species was necessary to model the enhanced rebinding below 30 K, which likely appears because of quantum tunneling. The peak positions and widths of the enthalpy distributions are similar for the P21 and P6 crystal forms, indicating that crystal-packing forces have only very minor effects on the structure at the active site. Moreover, the widths of the (dominant) distributions are qualitatively similar to those observed with glycerol-water solutions, which shows that the degree of structural heterogeneity is similar for solution and crystalline samples. For the A3 substate, a significantly lower peak enthalpy was obtained (by approximately 4 kJ/mol) than for solutions, while the peak shifts in the A0 and A1 substates were small. In samples cooled under illumination, discrete populations with higher rebinding barriers were observed. Concomitant changes in the stretch absorption of the photodissociated CO (B states) only occur between 100 and 130 K. They likely arise from movements of the ligand in the heme pocket between discrete sites.