Structure of a third cooperativity state of hemoglobin: ultraviolet resonance Raman spectroscopy of cyanomethemoglobin ligation microstates.

Ultraviolet resonance Raman (UVRR) spectra have been obtained for cyanometHb (Hb = hemoglobin) hybrid tetramers representing several ligation microstates, using dimer interchange techniques and spectral subtraction. Relative to fully ligated tetramers, Hb(CN)4, the monoligated hybrids and deoxyHb all show fully developed T/R difference UVRR bands which are associated with T state quaternary contacts across the alpha 1 beta 2 interface, involving the Trp beta 37 and Tyr alpha 42 residues. Triligated species show quite different signals, arising from the interior residues Trp alpha 14 and/or beta 15. From earlier studies, these Rdeoxy signals are attributed to E helix displacement toward the heme in deoxy subunits within R state tetramers, resulting in weakened Trp H-bonds. Asymmetric diligated hybrids, containing both ligands in the same dimer unit, show signals characteristic of the T quaternary contacts, but they are attenuated by 40%. An equilibrium mixture of T and R state molecules is ruled out by the absence of significantly strong Rdeoxy difference bands. Rather, the spectral attenuation is attributed to weakening of the T state contacts at the alpha 1 beta 2 interface. This interpretation is supported by previous observations that the mutational pattern of free energy perturbations for the asymmetric hybrid is T-like and not that of a T/R equilibrium or an R-like state. The asymmetric hybrid represents a third cooperativity state, T', having a T quaternary arrangement of the subunits but a deformed alpha 1 beta 2 interface, with weakened contacts.