Heme Reactivity is Uncoupled
from Quaternary Structure
in Gel-Encapsulated Hemoglobin: A Resonance Raman Spectroscopic Study
- Publication date
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Abstract
Encapsulation of hemoglobin (Hb) in silica gel preserves
structure
and function but greatly slows protein motion, thereby providing access
to intermediates along the allosteric pathway that are inaccessible
in solution. Resonance Raman (RR) spectroscopy with visible and ultraviolet
laser excitation provides probes of heme reactivity and of key tertiary
and quaternary contacts. These probes were monitored in gels after
deoxygenation of oxyHb and after CO binding to deoxyHb, which initiate
conformational change in the R–T and T–R directions,
respectively. The spectra establish that quaternary structure change
in the gel takes a week or more but that the evolution of heme reactivity,
as monitored by the Fe–histidine stretching vibration, ν<sub>FeHis</sub>, is completed within two days, and is therefore uncoupled
from the quaternary structure. Within each quaternary structure, the
evolving ν<sub>FeHis</sub> frequencies span the full range of
values between those previously associated with the high- and low-affinity
end states, R and T. This result supports the tertiary two-state (TTS)
model, in which the Hb subunits can adopt high- and low-affinity tertiary
structures, <i>r</i> and <i>t,</i> within each
quaternary state. The spectra also reveal different tertiary pathways,
involving the breaking and reformation of E and F interhelical contacts
in the R–T direction but not the T–R direction. In the
latter, tertiary motions are restricted by the T quaternary contacts