A CRYSTALLOGRAPHIC EXAMINATION OF HEMOGLOBIN LIGAND BINDING AND ALLOSTERY. Gregory B. Vásquez,1 Xinhua Ji,1 Igor Pechik,1 Michael Karavitis,2 Herman Kwansa,2 Enrico Bucci,2 Clara Fronticelli,2 and Gary L. Gilliland.1 1Center for Advanced Research in Biotechnology of the University of Maryland Biotechnology Institute and the National Institute of Standards and Technology, 9600 Gudelsky Drive, Rockville, MD 20850. 2Department of Biochemistry, University of Maryland at Baltimore, 108 N. Greene St., Baltimore, MD 21210.
Recent concerns about blood-borne pathogens, such as HIV and hepatitis, have accelerated studies of hemoglobin for the purpose of developing blood substitutes as an alternative to transfusions. Hemoglobin is probably one of the best studied allosteric proteins, but the mechanisms of its allosteric transformations (deoxy ' oxy or T ' R) are still elusive. In this study, two recombinant, mutant T-state hemoglobins (ßC112G and ß(C93A,C112G)) at 2.0 and 1.8 Å resolution, respectively, are reported along with the three-dimensional structure of R-state carbonmonoxy-hemoglobin at 2.2 Å resolution. The structures have been solved by difference Fourier techniques followed by least-squares refinement. The mutant ßC112G is partially oxygenated with oxygen bound to both a-hemes. The oxygen molecule associated with the a1 heme has full occupancy while the molecule associated with the a2 heme is partially occupied. The results of the comparison of this partially ligated, T-state hemoglobin with the structures of ß(C93A,C112G) and deoxyhemoglobin (Fronticelli, et al., J. Biol. Chem., 1994, 269, 23965-23969) indicate significant changes in the heme geometry and the position of the F-helix in the a1 subunit. In the partially ligated a2 subunit, only deviations in the positions of the iron and proximal histidine are observed. The R-state carbonmonoxyhemoglobin extends the structure from 2.7 Å (Baldwin, J. Mol. Biol. , 1980, 136, 103-128) to 2.2 Å resolution. The higher resolution structure has extensive solvent structure, including a sulfate binding site between both the a1ß2 and the a2ß1 subunits, and has Fe-C-O angles of 125deg. and 162deg. for the a and ß subunits, respectively, that differ from the angles previously reported in the lower resolution crystallographic study (Baldwin, 1980), however are nearer to the angles determined for myoglobin crystallographically (Kurijan, et al., J. Mol. Biol., 1986, 192, 133-154) and those determined spectroscopically (Lian, et al., Biochemistry, 1993, 32, 5809-5814; Schlichting, et al., Nature, 1994, 371, 808-812).