Structure-Function Relationships in Hemoglobin Kariya, Lys-40(C5)α → Glu, with High Oxygen Affinity: Functional role of the salt bridge between lys-40α and the β chain cooh terminus

Abstract

The ϵ-amino group of Lys-40α forms a salt bridge with the α-carboxyl group of β chain in deoxyhemoglobin and is considered to impose a constraint upon hemoglobin tetramer, stabilizing the T quaternary structure. Hb Kariya, in which Lys-40α is replaced by Glu, provides a unique opportunity to investigate the functional role of this salt bridge. Hb Kariya showed oxygen binding properties characterized by a high affinity, diminished cooperativity, a reduced alkaline Bohr effect, and a decreased effect of phosphates upon oxygen affinity. In deoxyHb Kariya the reactivity of the sulfhydryl groups of cysteins-93β with 4,4′-dipyridine disulfide was profoundly enhanced, being comparable to that for normal oxyhemoglobin (oxyHb A). The Soret band spectra, UV derivative spectra, and UV oxyminus-deoxy difference spectra indicated that oxyHb Kariya assumes a quaternary structure similar to that of oxyHb A whereas the T structure of deoxyHb Kariya is destabilized, and Hb Kariya remains predominantly in the R state upon deoxygenation. Resonance Raman scattering by deoxyHb Kariya showed that the Fe-Nϵ(proximal His) bond is less stretched than that of deoxyHb A. These experimental results provide structural basis for explaining the oxygen binding characteristics of Hb Kariya and further give direct evidence that the intersubunit salt bridge between Lys-40α and the β chain COOH terminus actually contributes to stabilization of the T quaternary structure, thereby playing a key role in cooperative oxygen binding by hemoglobin. The nature of another salt bridge between Asp-94β and the COOH-terminal His of β chain was also discussed in comparison with the salt bridge involving Lys-40α.