Hemoglobin allostery: resonance Raman spectroscopy of kinetic intermediates.

The end states, R and T, of the allosteric transition in hemoglobin (Hb) are structurally well characterized, but there is little information on intermediate structures along the allosteric pathway. These intermediates were examined by means of time-resolved resonance Raman spectroscopy in the nanosecond-to-microsecond interval after HbCO photolysis. Complementary spectra of the heme group and of the tyrosine and tryptophan residues were recorded during laser excitation at 436 and 230 nanometers. These spectra reveal a sequence of interleaved tertiary and quaternary motions during the photocycle, motions involving the proximal and distal helices, and the alpha 1 beta 2 subunit interface. This sequence leads to a modified form of the T state, in which the alpha 1 beta 2 interface is deformed as a result of two carbon monoxide molecules binding to the same dimer within the tetramer.