1 research outputs found
Differential Control of Heme Reactivity in Alpha and Beta Subunits of Hemoglobin: A Combined Raman Spectroscopic and Computational Study
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., <i>Biophys. Chem.</i> <b>2002</b>, <i>98</i>, 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., <i>Proc. Natl. Acad. Sci. U. S. A.</i> <b>2011</b>, <i>108</i>, 5608). The effector molecule IHP was found to lower
νFeHis selectively for α chains within the R state, and
a binding site in the α<sub>1</sub>α<sub>2</sub> cleft
is suggested