Site-directed mutagenesis in haemoglobin: Functional role of tyrosine-42(C7)α at the α1-β2 interface

To clarify the functional role of Tyr-42(C7)α, which forms a hydrogen bond with Asp-99(G1)β at the α1-β2 interface of human deoxyhaemoglobin, we engineered two artificial mutant haemoglobins (Hb), in which Tyr-42α was replaced by Phe (Hb Phe-42α) or His (Hb His-42α), and investigated their oxygen binding properties together with structural consequences of the mutations by using various spectroscopic probes. Like most of the natural Asp-99β mutants, Hb Phe-42α showed a markedly increased oxygen affinity, a reduced Bohr effect and diminished co-operativity. Structural probes such as ultraviolet-region derivative and oxy-minus-deoxy difference spectra, resonance Raman scattering and proton nuclear magnetic resonance spectra indicate that, in Hb Phe-42α, the deoxy T quaternary structure is highly destabilized and the strain imposed on the FeN ε (proximal His) bond is released, stabilizing the oxy tertiary structure. In contrast with Hb Phe-42α, Hb His-42α showed an intermediately impaired function and only moderate destabilization of the T-state, which can be explained by the formation of a new, weak hydrogen bond between His-42α and Asp-99β. Spectroscopic data were consistent with this assumption. The present study proves that the hydrogen bond between Tyr-42α and Asp-99β plays a key role in stabilizing the deoxy T structure and consequently in co-operative oxygen binding.