Quaternary structure sensitive tyrosine interactions in hemoglobin: A UV resonance raman study of the double mutant rHb (β99Asp→Asn, α42Tyr→Asp)

Shoucai Huang, Eric S. Peterson, Chien Ho, Joel M. Friedman

Research output: Contribution to journalArticle

47 Citations (Scopus)

Abstract

Two interactions involving tyrosines have been implicated in the communication pathway that links ligand binding to quaternary state changes in hemoglobin. Tyr α1 42 stabilizes the α12 T state interface through the formation of a hydrogen bond to Asp β299. The side chains of the penultimate Tyr residues (α140 and β145) occupy the pockets made by helicies F and H in the deoxy form with the phenolic hydroxyl hydrogen bonded to the carbonyl group of Val FG5. Early crystallographic studies indicated that in the R form the penultimate Tyr is expelled out of the pocket, thus eliminating the hydrogen bond. This hydrogen bond has been considered to play an important role in maintaining the low-oxygen-affinity state (T state) in deoxy HbA, but a later higher resolution crystallographic study (Shannon, 1983) failed to reveal such movement of this Tyr during the R → T transition. Nevertheless, conversion of this Tyr to Phe increases oxygen affinity considerably, suggesting that hydrogen bonding is involved in oxygen affinity modulation. Earlier ultraviolet resonance Raman results reported by Spiro and co-workers [Rodgers et al. (1992) J. Am. Chem. Soc. 114, 3697- 3709] were used to conclude that the significant quaternary structure dependent changes observed in tyrosine Raman bands are due to the formation of the T state hydrogen bond with Tyr α42 acting as a proton acceptor rather than being the anticipated proton donor, as would be expected if Asp β99 were ionized. This surprising result rests on the assumption that changes in the environment of Tyr α42 are the overwhelming contributor to the R - T UV Raman difference spectrum. In this study, a cooperative double mutant lacking Tyr α42, [rh (Asp β99 → Asn, Tyr α42 Asp)], is used to determine the relative contributions of Tyr α42 and the penultimate tyrosines to the R -T UV resonance Raman difference spectrum. The results both directly support the claim that Tyr α42 is the proton acceptor in the T state and expose the potential role of the penultimate tyrosines in coupling the quaternary state to the ligand reactivity.

Original languageEnglish (US)
Pages (from-to)6197-6206
Number of pages10
JournalBiochemistry
Volume36
Issue number20
DOIs
StatePublished - May 20 1997

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Tyrosine
Hydrogen
Hydrogen bonds
Hemoglobins
Protons
Oxygen
Ligands
Interface states
Hydrogen Bonding
Viperidae
Hydroxyl Radical
Modulation
Communication

ASJC Scopus subject areas

  • Biochemistry

Cite this

Quaternary structure sensitive tyrosine interactions in hemoglobin : A UV resonance raman study of the double mutant rHb (β99Asp→Asn, α42Tyr→Asp). / Huang, Shoucai; Peterson, Eric S.; Ho, Chien; Friedman, Joel M.

In: Biochemistry, Vol. 36, No. 20, 20.05.1997, p. 6197-6206.

Research output: Contribution to journalArticle

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abstract = "Two interactions involving tyrosines have been implicated in the communication pathway that links ligand binding to quaternary state changes in hemoglobin. Tyr α1 42 stabilizes the α1/β2 T state interface through the formation of a hydrogen bond to Asp β299. The side chains of the penultimate Tyr residues (α140 and β145) occupy the pockets made by helicies F and H in the deoxy form with the phenolic hydroxyl hydrogen bonded to the carbonyl group of Val FG5. Early crystallographic studies indicated that in the R form the penultimate Tyr is expelled out of the pocket, thus eliminating the hydrogen bond. This hydrogen bond has been considered to play an important role in maintaining the low-oxygen-affinity state (T state) in deoxy HbA, but a later higher resolution crystallographic study (Shannon, 1983) failed to reveal such movement of this Tyr during the R → T transition. Nevertheless, conversion of this Tyr to Phe increases oxygen affinity considerably, suggesting that hydrogen bonding is involved in oxygen affinity modulation. Earlier ultraviolet resonance Raman results reported by Spiro and co-workers [Rodgers et al. (1992) J. Am. Chem. Soc. 114, 3697- 3709] were used to conclude that the significant quaternary structure dependent changes observed in tyrosine Raman bands are due to the formation of the T state hydrogen bond with Tyr α42 acting as a proton acceptor rather than being the anticipated proton donor, as would be expected if Asp β99 were ionized. This surprising result rests on the assumption that changes in the environment of Tyr α42 are the overwhelming contributor to the R - T UV Raman difference spectrum. In this study, a cooperative double mutant lacking Tyr α42, [rh (Asp β99 → Asn, Tyr α42 Asp)], is used to determine the relative contributions of Tyr α42 and the penultimate tyrosines to the R -T UV resonance Raman difference spectrum. The results both directly support the claim that Tyr α42 is the proton acceptor in the T state and expose the potential role of the penultimate tyrosines in coupling the quaternary state to the ligand reactivity.",
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