Heterogeneity between Two α Subunits of α₂β₂ Human Hemoglobin and O₂ Binding Properties: Raman, ¹H Nuclear Magnetic Resonance, and Terahertz Spectra

Following a previous detailed investigation of the β subunit of α₂β₂ human adult hemoglobin (Hb A), this study focuses on the α subunit by using three natural valency hybrid α(Fe²⁺-deoxy/O₂)β(Fe³⁺) hemoglobin M (Hb M) in which O₂ cannot bind to the β subunit: Hb M Hyde Park (β92His → Tyr), Hb M Saskatoon (β63His → Tyr), and Hb M Milwaukee (β67Val → Glu). In contrast with the β subunit that exhibited a clear correlation between O₂ affinity and Fe²⁺-His stretching frequencies, the Fe²⁺-His stretching mode of the α subunit gave two Raman bands only in the T quaternary structure. This means the presence of two tertiary structures in α subunits of the α₂β₂ tetramer with T structure, and the two structures seemed to be nondynamical as judged from terahertz absorption spectra in the 5-30 cm^-1 region of Hb M Milwaukee, α(Fe²⁺-deoxy)β(Fe³⁺). This kind of heterogeneity of α subunits was noticed in the reported spectra of a metal hybrid Hb A like α(Fe²⁺-deoxy)β(Co²⁺) and, therefore, seems to be universal among α subunits of Hb A. Unexpectedly, the two Fe-His frequencies were hardly changed with a large alteration of O₂ affinity by pH change, suggesting no correlation of frequency with O₂ affinity for the α subunit. Instead, a new Fe²⁺-His band corresponding to the R quaternary structure appeared at a higher frequency and was intensified as the O₂ affinity increased. The high-frequency counterpart was also observed for a partially O₂-bound form, α(Fe²⁺-deoxy)α(Fe²⁺-O₂)β(Fe³⁺)β(Fe³⁺), of the present Hb M, consistent with our previous finding that binding of O₂ to one α subunit of T structure α₂β₂ tetramer changes the other α subunit to the R structure.