Limits...
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.

Show MeSH

Related in: MedlinePlus

Pump–probe differencespectra at the indicated delay times,reflecting alterations in tertiary (0.15 μs) and quaternary(3 and 50 μs) H-bonds,10 which revealthe same dynamics for HbA and the Hb hybrids.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4353013&req=5

fig2: Pump–probe differencespectra at the indicated delay times,reflecting alterations in tertiary (0.15 μs) and quaternary(3 and 50 μs) H-bonds,10 which revealthe same dynamics for HbA and the Hb hybrids.

Mentions: Pump–probe UVrR difference spectroscopyhas shown that thesequaternary contacts are formed in two successive time steps followingligand photodissociation from HbCO, first the hinge (2 μs) andthen the switch (20 μs).10 We found(Figure 2) that the difference spectra wereidentical for the hybrids and HbA at 3.0 and 50 μs, before andafter the switch contact is formed. Likewise identical spectra wereobtained at 0.15 μs, following the first detectable tertiarytransition (0.07 μs)10,70−72 and before the quaternary movements. This difference spectrum isassociated with the breaking of interhelical H-bonds due to the concertedmotion of the E and F helices.33,72,73


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)

Pump–probe differencespectra at the indicated delay times,reflecting alterations in tertiary (0.15 μs) and quaternary(3 and 50 μs) H-bonds,10 which revealthe same dynamics for HbA and the Hb hybrids.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Pump–probe differencespectra at the indicated delay times,reflecting alterations in tertiary (0.15 μs) and quaternary(3 and 50 μs) H-bonds,10 which revealthe same dynamics for HbA and the Hb hybrids.
Mentions: Pump–probe UVrR difference spectroscopyhas shown that thesequaternary contacts are formed in two successive time steps followingligand photodissociation from HbCO, first the hinge (2 μs) andthen the switch (20 μs).10 We found(Figure 2) that the difference spectra wereidentical for the hybrids and HbA at 3.0 and 50 μs, before andafter the switch contact is formed. Likewise identical spectra wereobtained at 0.15 μs, following the first detectable tertiarytransition (0.07 μs)10,70−72 and before the quaternary movements. This difference spectrum isassociated with the breaking of interhelical H-bonds due to the concertedmotion of the E and F helices.33,72,73

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