ATOMIC RESOLUTION STRUCTURE OF DEOXY-MYOGLOBIN AT 293K AND 100K
Galina S. Kachalova1, Alexander N. Popov1,2, and Hans D. Bartunik1
1 Max-Planck-Arbeitsgruppe
Proteindynamik, MPG-ASMB, c/o DESY, Notkestrasse 85, 22607,
Hamburg, Germany
2 Present adress: EMBL, c/o DESY,Notkestrasse 85, 22607,
Hamburg, Germany.
Keywords: Heme protein; protein crystallography; ultra-high resolution; synchrotron radiation.
Investigations of motions in heme proteins that may be induced by ligand binding or photolysis require detailed knowledge of the conformation of their deoxy forms as the ground states, in particular of the heme geometry, the relative orientations of neighbouring protein residues, and of the protein matrix. Previous crystal structure analyses of a number of different heme proteins in their deoxy states led to controversial results, partly due to preparational problems causing contaminations with different ligandation states and to insufficient resolution. As part of an investigation of ligand-induced conformational changes in myoglobin at true atomic resolution, we prepared Mb in its deoxy state by a new technique and studied its structure at RT (293K) and at 100K. The diffraction data were measured on BW6/DORIS. The structures were refined at 0.9 A (100K) and 1.15 A resolution (293K), respectively, with the program SHELXL-96 [1] employing restrained anisotropic displacement parameters.
We obtained density maps of high contrast. In both structures, a water molecule is located at the same unique position ('deoxy-water site') near the sixth coordination site of the iron. This deoxy water has partial occupancy and interacts with NE2 of the distal histidine, His 64, and with the NC atom of the porphyrin. The relative positions and orientations of the heme and of neighbouring residues are identical in the two structures, despite changes in the intermolecular contact regions upon shock-freezing. In both structures, His 64 is present in two well-defined alternate sites. The positions of the iron atom relative to the heme plane in the two structures agree within 0.01 A, indicating the high accuracy in the refined structures.