Heterogeneity between Two α Subunits of α 2 β 2 Human Hemoglobin and O 2 Binding Properties: Raman, 1 H Nuclear Magnetic Resonance, and Terahertz Spectra.

Following a previous detailed investigation of the β subunit of α ₂ β ₂ human adult hemoglobin (Hb A), this study focuses on the α subunit by using three natural valency hybrid α(Fe ²⁺ -deoxy/O ₂ )β(Fe ³⁺ ) hemoglobin M (Hb M) in which O ₂ 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 O ₂ affinity and Fe ²⁺ -His stretching frequencies, the Fe ²⁺ -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 α ₂ β ₂ 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(Fe ²⁺ -deoxy)β(Fe ³⁺ ). This kind of heterogeneity of α subunits was noticed in the reported spectra of a metal hybrid Hb A like α(Fe ²⁺ -deoxy)β(Co ²⁺ ) 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 O ₂ affinity by pH change, suggesting no correlation of frequency with O ₂ affinity for the α subunit. Instead, a new Fe ²⁺ -His band corresponding to the R quaternary structure appeared at a higher frequency and was intensified as the O ₂ affinity increased. The high-frequency counterpart was also observed for a partially O ₂ -bound form, α(Fe ²⁺ -deoxy)α(Fe ²⁺ -O ₂ )β(Fe ³⁺ )β(Fe ³⁺ ), of the present Hb M, consistent with our previous finding that binding of O ₂ to one α subunit of T structure α ₂ β ₂ tetramer changes the other α subunit to the R structure.