The conformational and dynamic basis for ligand binding reactivity in hemoglobin Ypsilanti (beta 99 asp -> Tyr): Origin of the quaternary enhancementeffects

Hemoglobin Ypsilanti (HbY) is a stable tetrameric hemoglobin that binds oxy gen with little or no cooperativity and with high affinity [Doyle, M. L., e t al. (1992) Proteins: Struct., Funct., Genet. 14, 351-362]. It displays an especially large quaternary enhancement effect. An X-ray crystallographic study [Smith, F. R., et al. (1991) Proteins: Struct., Funct., Genet. 10, 81 -91] of the carboxy derivative of this hemoglobin (COHbY) revealed a new qu aternary structure that partially resembles the recently described R-2 stru cture [Silva, M. M., et al. (1992) J. Biol. Chem. 267, 17248-17256]. Very l ittle is known about either the solution phase conformations of the ligande d and deoxy forms of HbY or the molecular basis for the large quaternary en hancement effect (Doyle et al., 1992). In this study, near-IR absorption, S oret-enhanced Raman, and UV (229 nm) resonance Raman spectroscopies are use d to probe the liganded and deoxy derivatives of HbY in solution. Nanosecon d time-resolved near-IR absorption measurements are used to expose the rela xation properties of the photoproduct of COHbY. Time-resolved (Soret band) absorption is used to generate the geminate and solvent phase ligand rebind ing curves for photodissociated COHbY. The spectroscopic results indicate t hat COHbY has an R-like conformation with respect to both the proximal heme pocket and the hinge region of the alpha(1)beta(2) interface. The deoxy de rivative of HbY has spectroscopic features that are very similar to those o bserved for species assigned to the deoxy R or half-liganded R conformation s of human adult hemoglobin (HbA). The 10 ns to 100 mu s relaxation propert ies of the photoproduct of COHbY are distinctly different from those of HbA in that for HbY, little if any tertiary or quaternary relaxation is observ ed. The near-absence of relaxation in the HbY photoproduct explains the dif ferences in the geminate and solvent phase CO recombination between HbA and HbY. The impact of the conformational and relaxation properties of HbY on the geminate rebinding process forms the basis of a model that accounts for the large quaternary enhancement effect reported for HbY (Doyle et al., 19 92). In addition, the spectroscopic data and the X-ray crystallographic res ults explain the slow relaxation for HbY and the near-absence of cooperativ e ligand binding for this protein based on the behavior of the penultimate tyrosines.