Towards understanding non-equivalence of α and β subunits within human hemoglobin in conformational relaxation and molecular oxygen rebinding.

Picosecond to millisecond laser time-resolved transient absorption spectroscopy was used to study molecular oxygen (O2) rebinding and conformational relaxation following O2 photodissociation in the α and β subunits within human hemoglobin in the quaternary R-like structure. Oxy-cyanomet valency hybrids, α2(Fe2+-O2)β2(Fe3+-CN) and α2(Fe3+-CN)β2(Fe2+-O2), were used as models for oxygenated R-state hemoglobin. An extended kinetic model for geminate O2 rebinding in the ferrous hemoglobin subunits, ligand migration between the primary and secondary docking site(s), and nonexponential tertiary relaxation within the R quaternary structure, was introduced and discussed. Significant functional non-equivalence of the α and β subunits in both the geminate O2 rebinding and concomitant structural relaxation was revealed. For the β subunits, the rate constant for the geminate O2 rebinding to the unrelaxed tertiary structure and the tertiary transition rate were found to be greater than the corresponding values for the α subunits. The conformational relaxation following the O2 photodissociation in the α and β subunits was found to decrease the rate constant for the geminate O2 rebinding, this effect being more than one order of magnitude greater for the β subunits than for the α subunits. Evidence was provided for the modulation of the O2 rebinding to the individual α and β subunits within human hemoglobin in the R-state structure by the intrinsic heme reactivity through a change in proximal constraints upon the relaxation of the tertiary structure on a picosecond to microsecond time scale. Our results demonstrate that, for native R-state oxyhemoglobin, O2 rebinding properties and spectral changes following the O2 photodissociation can be adequately described as the sum of those for the α and β subunits within the valency hybrids. The isolated β chains (hemoglobin H) show similar behavior to the β subunits within the valency hybrids and can be used as a model for the β subunits within the R-state oxyhemoglobin. At the same time, the isolated α chains behave differently to the α subunits within the valency hybrids.