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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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νFeHis (filled circles) and CO saturation (open circles)in HbCO (black) and MbCO (red) solutions, as functions of the incident16 ns laser pulse energy.
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fig4: νFeHis (filled circles) and CO saturation (open circles)in HbCO (black) and MbCO (red) solutions, as functions of the incident16 ns laser pulse energy.

Mentions: However, the reportedfrequency of this prompt signal has been somewhat variable. We discoveredthat one reason for this variability is that the measured frequencydecreases with the increasing incident laser power. Figure 4 shows the power-dependent νFeHis values obtainedfrom rR spectra produced by our 16 ns laser pulses (which act as simultaneouspump and probe). At low power levels only a fraction of the HbCO isphotodissociated; this fraction (calculated from the normalized intensityratio of the separate ν4 porphyrin bands of deoxyand CO-heme9,80) is also plotted in Figure 4. The νFeHis values were determined by Guassianpeak fitting, after subtraction of the CO-heme spectrum (there isa weak 238 cm–1 CO-heme band), using the isolated508 cm–1 Fe–CO stretching band81 for normalization. At 1 μJ/pulse, wherethe photolyzed fraction is <5%, νFeHis = 228.5 cm–1, but drops rapidly to a plateau value of 225.5 cm–1 beyond 5 μJ/pulse. Importantly, this effect is much weakerfor myoglobin (Mb), which shows only a 1 cm–1 initialdrop, to 220.5 cm–1, which is the equilibrium deoxyMbvalue. An even larger νFeHis difference between Hb and Mb wasreported by Mizutani and Nagai,82,83 using picosecond pump–probespectroscopy. A few ps after HbCO photolysis, νFeHis was 232cm–1 for Hb, while for Mb it was 221 cm–1.


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)

νFeHis (filled circles) and CO saturation (open circles)in HbCO (black) and MbCO (red) solutions, as functions of the incident16 ns laser pulse energy.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: νFeHis (filled circles) and CO saturation (open circles)in HbCO (black) and MbCO (red) solutions, as functions of the incident16 ns laser pulse energy.
Mentions: However, the reportedfrequency of this prompt signal has been somewhat variable. We discoveredthat one reason for this variability is that the measured frequencydecreases with the increasing incident laser power. Figure 4 shows the power-dependent νFeHis values obtainedfrom rR spectra produced by our 16 ns laser pulses (which act as simultaneouspump and probe). At low power levels only a fraction of the HbCO isphotodissociated; this fraction (calculated from the normalized intensityratio of the separate ν4 porphyrin bands of deoxyand CO-heme9,80) is also plotted in Figure 4. The νFeHis values were determined by Guassianpeak fitting, after subtraction of the CO-heme spectrum (there isa weak 238 cm–1 CO-heme band), using the isolated508 cm–1 Fe–CO stretching band81 for normalization. At 1 μJ/pulse, wherethe photolyzed fraction is <5%, νFeHis = 228.5 cm–1, but drops rapidly to a plateau value of 225.5 cm–1 beyond 5 μJ/pulse. Importantly, this effect is much weakerfor myoglobin (Mb), which shows only a 1 cm–1 initialdrop, to 220.5 cm–1, which is the equilibrium deoxyMbvalue. An even larger νFeHis difference between Hb and Mb wasreported by Mizutani and Nagai,82,83 using picosecond pump–probespectroscopy. A few ps after HbCO photolysis, νFeHis was 232cm–1 for Hb, while for Mb it was 221 cm–1.

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