title: Site-directed mutagenesis in haemoglobin. Functional role of tyrosine-42(C7) alpha at the alpha 1-beta 2 interface.
abstract: To clarify the functional role of Tyr-42(C7) alpha, which forms a hydrogen bond with Asp-99(G1) beta at the alpha 1-beta 2 interface of human deoxyhaemoglobin, we engineered two artificial mutant haemoglobins (Hb), in which Tyr-42 alpha was replaced by Phe (Hb Phe-42 alpha) or His (Hb His-42 alpha), and investigated their oxygen binding properties together with structural consequences of the mutations by using various spectroscopic probes. Like most of the natural Asp-99 beta mutants, Hb Phe-42 alpha 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-42 alpha, the deoxy T quaternary structure is highly destabilized and the strain imposed on the Fe-N epsilon (proximal His) bond is released, stabilizing the oxy tertiary structure. In contrast with Hb Phe-42 alpha, Hb His-42 alpha 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-42 alpha and Asp-99 beta. Spectroscopic data were consistent with this assumption. The present study proves that the hydrogen bond between Tyr-42 alpha and Asp-99 beta plays a key role in stabilizing the deoxy T structure and consequently in co-operative oxygen binding.
authors.affiliation: Department of Physiochemical Physiology Medical School, Osaka University, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Imai
authors.forename: K
authors.initials: K
authors.lastname: Fushitani
authors.forename: G
authors.initials: G
authors.lastname: Miyazaki
authors.forename: K
authors.initials: K
authors.lastname: Ishimori
authors.forename: T
authors.initials: T
authors.lastname: Kitagawa
authors.forename: Y
authors.initials: Y
authors.lastname: Wada
authors.forename: H
authors.initials: H
authors.lastname: Morimoto
authors.forename: I
authors.initials: I
authors.lastname: Morishima
authors.forename: D T
authors.initials: DT
authors.lastname: Shih
authors.forename: J
authors.initials: J
authors.lastname: Tame
mesh_terms: D006454:Hemoglobins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy; D016297:Mutagenesis, Site-Directed; D010100:Oxygen / Q000378:metabolism; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013055:Spectrophotometry, Infrared; D013056:Spectrophotometry, Ultraviolet; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D006454:Hemoglobins; D014443:Tyrosine; D010100:Oxygen