title: Differential control of heme reactivity in alpha and beta subunits of hemoglobin: a combined Raman spectroscopic and computational study.
abstract: The use of hybrid hemoglobin (Hb), with mesoheme substituted for protoheme, allows separate monitoring of the α or β hemes along the allosteric pathway. Using resonance Raman (rR) spectroscopy in silica gel, which greatly slows protein motions, we have observed that the Fe-histidine stretching frequency, νFeHis, which is a monitor of heme reactivity, evolves between frequencies characteristic of the R and T states, for both α or β chains, prior to the quaternary R-T and T-R shifts. Computation of νFeHis, using QM/MM and the conformational search program PELE, produced remarkable agreement with experiment. Analysis of the PELE structures showed that the νFeHis shifts resulted from heme distortion and, in the α chain, Fe-His bond tilting. These results support the tertiary two-state model of ligand binding (Henry et al., Biophys. Chem. 2002, 98, 149). Experimentally, the νFeHis evolution is faster for β than for α chains, and pump-probe rR spectroscopy in solution reveals an inflection in the νFeHis time course at 3 μs for β but not for α hemes, an interval previously shown to be the first step in the R-T transition. In the α chain νFeHis dropped sharply at 20 μs, the final step in the R-T transition. The time courses are fully consistent with recent computational mapping of the R-T transition via conjugate peak refinement by Karplus and co-workers (Fischer et al., Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 5608). The effector molecule IHP was found to lower νFeHis selectively for α chains within the R state, and a binding site in the α1α2 cleft is suggested.
authors.affiliation: Department of Chemistry, University of Washington , Box 351700, Seattle, Washington 98195-1700, United States.
authors.forename: Eric M
authors.initials: EM
authors.lastname: Jones
authors.forename: Emanuele
authors.initials: E
authors.lastname: Monza
authors.forename: Gurusamy
authors.initials: G
authors.lastname: Balakrishnan
authors.forename: George C
authors.initials: GC
authors.lastname: Blouin
authors.forename: Piotr J
authors.initials: PJ
authors.lastname: Mak
authors.forename: Qianhong
authors.initials: Q
authors.lastname: Zhu
authors.forename: James R
authors.initials: JR
authors.lastname: Kincaid
authors.forename: Victor
authors.initials: V
authors.lastname: Guallar
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Spiro
mesh_terms: D000494:Allosteric Regulation; D019295:Computational Biology / Q000379:methods*; D006418:Heme / Q000737:chemistry*; D006441:Hemoglobin A / Q000737:chemistry*; D008652:Mesoporphyrins / Q000737:chemistry; D008958:Models, Molecular; D020836:Protein Structure, Quaternary; D013059:Spectrum Analysis, Raman / Q000379:methods*; D055542:alpha-Globins / Q000737:chemistry*; D055544:beta-Globins / Q000737:chemistry*
chemical_list: D008652:Mesoporphyrins; D055542:alpha-Globins; D055544:beta-Globins; D006418:Heme; C031468:mesoporphyrin IX; D006441:Hemoglobin A