STRUCTURAL HETEROGENEITY AND LIGAND-BINDING IN CARBONMONOXY MYOGLOBINCRYSTALS AT CRYOGENIC TEMPERATURES

We have characterized the ligand-rebinding behavior of single crystal native sperm whale carbonmonoxy myoglobin (swMbCO) (space group P2(1)) and a synthetic mutant swMbCO (space group P6) at cryogenic temperatu res 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 s hort-flash and extended illumination protocols were performed. The TDS analysis yields the enthalpy barrier distributions for recombination in the individual taxonomic (A) substates, A(0), A(1), and A(3). A sin gle gaussian barrier distribution gave a good first-order description but was insufficient to precisely fit the data within each substate. A n additional minority species was necessary to model the enhanced rebi nding below 30 K, which likely appears because of quantum tunneling. T he peak positions and widths of the enthalpy distributions are similar for the P2(1) and P6 crystal forms, indicating that crystal-packing f orces have only very minor effects on the structure at the active site . Moreover, the widths of the (dominant) distributions are qualitative ly similar to those observed with glycerol-water solutions, which show s that the degree of structural heterogeneity is similar for solution and crystalline samples. For the A(3) substate, a significantly lower peak enthalpy was obtained (by similar to 4 kJ/mol) than for solutions , while the peak shifts in the A(0) and A(1) substates were small. In samples cooled under illumination, discrete populations with higher re binding barriers were observed. Concomitant changes in the stretch abs orption of the photodissociated CO (B states) only occur between 100 a nd 130 K. They likely arise from movements of the ligand in the heme p ocket between discrete sites.