X-RAY-DIFFRACTION STUDY OF DI-LIGATED AND TETRA-LIGATED T-STATE HEMOGLOBIN FROM HIGH SALT CRYSTALS

X-ray diffraction difference electron density maps at 3 Å resolution obtained from di and tetra-ligated T-state hemoglobin (Hb) crystals are reported. Crystals isomorphous with native deoxyhemoglobin were obtained from ammonium sulfate solutions incubated with the synthetic allosteric effector RSR-56. RSR-56 binds at two symmetry-related Hb central water cavity sites and each molecule has major interactions with three different subunit side-chains; one effector with Arg141α2 HC3, Lys99α1 G6 and Asn108β1 and the other with the symmetry related residues, Arg141α1 Lys99α2 and Asn108β2. Crystals mounted in a nitrogen filled glove box were di-ligated as previously found with polyethyleneglycol Hb crystals. Crystals mounted in air under a layer of mother liquor were bright red and showed all four heme groups ligated. The difference electron density from the di-ligated crystals showed atomic movements to be restricted to the immediate neighborhood of the heme groups and the allosteric effector. By contrast, the tetra-ligated structure showed extended difference electron density near amino acid residues around both α and β heme groups and along the α1 β2 interface. Ligation of the β heme group appears to magnify the difference density around the α heme groups. There is no evidence of breakage of the Bohr salt bridge, His146β HC3 → Asp94β FG1, in the crystal. The observed difference electron density maps may help to clarify the way the allosteric mechanism is triggered. © 1992.