Site-directed mutagenesis in hemoglobin: functional and structural study of the intersubunit hydrogen bond of threonine-38(C3)alpha at the alpha 1-beta 2 interface in human hemoglobin.
To clarify the functional and structural roles of Thr-38 alpha at the alpha 1-beta 2 interface, two artificial alpha-chain mutants, in which Thr-38 alpha is replaced by Ser (Hb T38 alpha S) or Val (Hb T38 alpha V), were prepared. Thr-38 alpha is one of the highly conserved amino acid residues in hemoglobins and forms a hydrogen bond to Asp-99 beta, which is a crucial residue to stabilize the T state, via a water molecule in the deoxygenated form. We investigated their oxygen binding properties together with structural consequences of the mutations by using various spectroscopic probes. Their oxygen equilibrium curves showed small changes in the oxygen binding properties. Structural probes such as ultraviolet-region derivative and oxy-minus-deoxy difference spectra, resonance Raman scattering, and 1H-NMR spectra also indicated that the oxy and deoxy forms of these mutants show spectra characteristic of the R and T states, respectively, and the R-T transition is not very disturbed. The present structural and functional data of the mutants imply that the hydrogen bond between Thr-38 alpha and Asp-99 beta does not play a key role in stabilizing the deoxy T structure, which is in sharp contrast to the role of the hydrogen bond between Tyr-42 alpha and Asp-99 beta, and suggest that the interactions via the intersubunit hydrogen bonds are highly site-specific, depending on the amino acid residue which participates in them.
Hashimoto
M
M
Division of Molecular Engineering, Graduate School of Engineering, Kyoto University, Japan.
Ishimori
K
K
Imai
K
K
Miyazaki
G
G
Morimoto
H
H
Wada
Y
Y
Morishima
I
I
D005914:Globins / Q000737:chemistry / Q000235:genetics; D006441:Hemoglobin A / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism; D010782:Photolysis; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman; D013912:Threonine / Q000737:chemistry*
D013912:Threonine; D005914:Globins; D006441:Hemoglobin A; D010100:Oxygen