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J Am Chem Soc. 2014 Jul 23;136(29):10325-39. doi: 10.1021/ja503328a. Epub 2014 Jul 14.

Differential control of heme reactivity in alpha and beta subunits of hemoglobin: a combined Raman spectroscopic and computational study.

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Department of Chemistry, University of Washington , Box 351700, Seattle, Washington 98195-1700, United States.


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.

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