STRUCTURE OF A 3RD COOPERATIVITY STATE OF HEMOGLOBIN - ULTRAVIOLET RESONANCE RAMAN-SPECTROSCOPY OF CYANOMETHEMOGLOBIN LIGATION MICROSTATES

Ultraviolet resonance Raman (UVRR) spectra have been obtained for cyan ometHb (Hb = hemoglobin) hybrid tetramers representing several ligatio n microstates, using dimer interchange techniques and spectral subtrac tion. Relative to fully ligated tetramers, Hb(CN)(4), the monoligated hybrids and deoxyHb all show fully developed Tm 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 resi dues. Triligated species show quite different signals, arising from th e interior residues Trp alpha 14 and/or beta 15. From earlier studies, these R(deoxy) signals are attributed to E helix displacement toward the heme in deoxy subunits within R state tetramers, resulting in weak ened Trp H-bonds. Asymmetric diligated hybrids, containing both ligand s in the same dimer unit, show signals characteristic of the T quatern ary contacts, but they are attenuated by 40%. An equilibrium mixture o f T and R state molecules is ruled out by the absence of significantly strong R(deoxy) 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 observation s that the mutational pattern of free energy perturbations for the asy mmetric hybrid is T-like and not that of a T/R equilibrium or an R-lik e 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.