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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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Related in: MedlinePlus

Time coursesfor protoheme νFe–His in the αpβm (top panel) and αmβp (bottompanel) hybrid Hbs following addition of CO to deoxyHbgels. The addition of IHP (open squares) has only a minor effect,as shown. The insets are examples of rR νFeHis peaks for selected-IHP time points. Fitted time courses are shown as solid lines. (Thefirst αpβm time point was excludedfrom the fit.).
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fig5: Time coursesfor protoheme νFe–His in the αpβm (top panel) and αmβp (bottompanel) hybrid Hbs following addition of CO to deoxyHbgels. The addition of IHP (open squares) has only a minor effect,as shown. The insets are examples of rR νFeHis peaks for selected-IHP time points. Fitted time courses are shown as solid lines. (Thefirst αpβm time point was excludedfrom the fit.).

Mentions: The T–R direction (Figure 5) is inducedin gels by adding CO to encapsulated deoxyHb just prior to data collection.The rR probe beam transiently photodissociates bound CO to producethe five-coordinate heme complex required for νFeHis detection.Consequently, the 16 ns, 5 μJ laser pulses used in this experimentprobe unrelaxed heme in the photoproduct, as discussed above, andtherefore, the measured νFeHis values are expected to be elevatedfrom those seen in the R–T gels. If roughly 6 to 7 cm–1 is subtracted from the T–R frequencies, then νFeHisevolution is seen to be just the reverse of the R–T direction(Figure 3), with α chains starting outat somewhat lower frequencies than β chains in deoxyHb, andconverging to similar R state deoxy frequencies. These results aremirrored in encapsulated native Hb,17 whereνFeHis rises to values characteristic of the R state photoproduct(measured in HbCO gel) within 2 days. In both hybrids and native Hb,these events occur long before the full establishment of the UVrRmarkers of the T–R quaternary transition.17


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 coursesfor protoheme νFe–His in the αpβm (top panel) and αmβp (bottompanel) hybrid Hbs following addition of CO to deoxyHbgels. The addition of IHP (open squares) has only a minor effect,as shown. The insets are examples of rR νFeHis peaks for selected-IHP time points. Fitted time courses are shown as solid lines. (Thefirst αpβm time point was excludedfrom the fit.).
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Time coursesfor protoheme νFe–His in the αpβm (top panel) and αmβp (bottompanel) hybrid Hbs following addition of CO to deoxyHbgels. The addition of IHP (open squares) has only a minor effect,as shown. The insets are examples of rR νFeHis peaks for selected-IHP time points. Fitted time courses are shown as solid lines. (Thefirst αpβm time point was excludedfrom the fit.).
Mentions: The T–R direction (Figure 5) is inducedin gels by adding CO to encapsulated deoxyHb just prior to data collection.The rR probe beam transiently photodissociates bound CO to producethe five-coordinate heme complex required for νFeHis detection.Consequently, the 16 ns, 5 μJ laser pulses used in this experimentprobe unrelaxed heme in the photoproduct, as discussed above, andtherefore, the measured νFeHis values are expected to be elevatedfrom those seen in the R–T gels. If roughly 6 to 7 cm–1 is subtracted from the T–R frequencies, then νFeHisevolution is seen to be just the reverse of the R–T direction(Figure 3), with α chains starting outat somewhat lower frequencies than β chains in deoxyHb, andconverging to similar R state deoxy frequencies. These results aremirrored in encapsulated native Hb,17 whereνFeHis rises to values characteristic of the R state photoproduct(measured in HbCO gel) within 2 days. In both hybrids and native Hb,these events occur long before the full establishment of the UVrRmarkers of the T–R quaternary transition.17

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