Heme reactivity is uncoupled from quaternary structure in gel-encapsulated hemoglobin: a resonance Raman spectroscopic study

J Am Chem Soc. 2012 Feb 22;134(7):3461-71. doi: 10.1021/ja210126j. Epub 2012 Feb 9.

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(FeHis), is completed within two days, and is therefore uncoupled from the quaternary structure. Within each quaternary structure, the evolving ν(FeHis) 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, r and t, 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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Carbon Monoxide / metabolism
  • Heme / chemistry
  • Heme / metabolism*
  • Hemoglobin A / chemistry
  • Hemoglobin A / metabolism*
  • Humans
  • Immobilized Proteins / chemistry
  • Immobilized Proteins / metabolism*
  • Models, Molecular
  • Protein Structure, Tertiary
  • Spectrum Analysis, Raman / methods*

Substances

  • Immobilized Proteins
  • Heme
  • Carbon Monoxide
  • Hemoglobin A