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

Jones EM, Monza E, Balakrishnan G, Blouin GC, Mak PJ, Zhu Q, Kincaid JR, Guallar V, Spiro TG - J. Am. Chem. Soc. (2014)

Bottom Line: Natl.Acad.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.

View Article: PubMed Central - PubMed

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

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.

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Time courses for νFeHis for the indicated subunit containingprotoheme in the αpβm (black) andαmβp (red) hybrid Hbs, followingHbO2 deoxygenation in gels (top) or HbCO photodissociationin solution (bottom). The effect of IHP addition (open squares) ingels is also shown. Time courses for HbA (αpβp) under the same conditions are given as dashed lines forcomparison. Dotted horizontal lines indicate frequencies for unrelaxeddeoxy heme, after photolysis of HbO2 in gels, or of HbCOin solution (bottom). Marked at right are the chain frequencies fordeoxyHb in gels (top) and solution (bottom). Dotted vertical linesare labeled along the bottom axis showing time constants for tertiaryand quaternary contact changes.
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fig3: Time courses for νFeHis for the indicated subunit containingprotoheme in the αpβm (black) andαmβp (red) hybrid Hbs, followingHbO2 deoxygenation in gels (top) or HbCO photodissociationin solution (bottom). The effect of IHP addition (open squares) ingels is also shown. Time courses for HbA (αpβp) under the same conditions are given as dashed lines forcomparison. Dotted horizontal lines indicate frequencies for unrelaxeddeoxy heme, after photolysis of HbO2 in gels, or of HbCOin solution (bottom). Marked at right are the chain frequencies fordeoxyHb in gels (top) and solution (bottom). Dotted vertical linesare labeled along the bottom axis showing time constants for tertiaryand quaternary contact changes.

Mentions: The spectral datawere processed with Grams/AI (7.0) software (ThermoGalactic). The νFeHis band near 220 cm–1 wasfit with a Gaussian peak to obtain the center frequency. Examplesof the νFeHis spectra are presented in reference9 for seven of the 20 pump–probe delay times plottedin Figure 3. The time dependence of these deconvolutedFe–His stretching frequencies was modeled by a three-exponentialfunction with fixed time constants obtained from UVRR spectroscopy,which tracks both tertiary and quaternary changes.


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

Jones EM, Monza E, Balakrishnan G, Blouin GC, Mak PJ, Zhu Q, Kincaid JR, Guallar V, Spiro TG - J. Am. Chem. Soc. (2014)

Time courses for νFeHis for the indicated subunit containingprotoheme in the αpβm (black) andαmβp (red) hybrid Hbs, followingHbO2 deoxygenation in gels (top) or HbCO photodissociationin solution (bottom). The effect of IHP addition (open squares) ingels is also shown. Time courses for HbA (αpβp) under the same conditions are given as dashed lines forcomparison. Dotted horizontal lines indicate frequencies for unrelaxeddeoxy heme, after photolysis of HbO2 in gels, or of HbCOin solution (bottom). Marked at right are the chain frequencies fordeoxyHb in gels (top) and solution (bottom). Dotted vertical linesare labeled along the bottom axis showing time constants for tertiaryand quaternary contact changes.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4353013&req=5

fig3: Time courses for νFeHis for the indicated subunit containingprotoheme in the αpβm (black) andαmβp (red) hybrid Hbs, followingHbO2 deoxygenation in gels (top) or HbCO photodissociationin solution (bottom). The effect of IHP addition (open squares) ingels is also shown. Time courses for HbA (αpβp) under the same conditions are given as dashed lines forcomparison. Dotted horizontal lines indicate frequencies for unrelaxeddeoxy heme, after photolysis of HbO2 in gels, or of HbCOin solution (bottom). Marked at right are the chain frequencies fordeoxyHb in gels (top) and solution (bottom). Dotted vertical linesare labeled along the bottom axis showing time constants for tertiaryand quaternary contact changes.
Mentions: The spectral datawere processed with Grams/AI (7.0) software (ThermoGalactic). The νFeHis band near 220 cm–1 wasfit with a Gaussian peak to obtain the center frequency. Examplesof the νFeHis spectra are presented in reference9 for seven of the 20 pump–probe delay times plottedin Figure 3. The time dependence of these deconvolutedFe–His stretching frequencies was modeled by a three-exponentialfunction with fixed time constants obtained from UVRR spectroscopy,which tracks both tertiary and quaternary changes.

Bottom Line: Natl.Acad.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.

View Article: PubMed Central - PubMed

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

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.

Show MeSH
Related in: MedlinePlus