Quaternary structure sensitive tyrosine interactions in hemoglobin: a UV resonance Raman study of the double mutant rHb (beta99Asp-->Asn, alpha42Tyr-->Asp).
Two interactions involving tyrosines have been implicated in the communication pathway that links ligand binding to quaternary state changes in hemoglobin. Tyr alpha(1)42 stabilizes the alpha1beta2 T state interface through the formation of a hydrogen bond to Asp beta(2)99. The side chains of the penultimate Tyr residues (alpha140 and beta145) 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 alpha42 acting as a proton acceptor rather than being the anticipated proton donor, as would be expected if Asp beta99 were ionized. This surprising result rests on the assumption that changes in the environment of Tyr alpha42 are the overwhelming contributor to the R - T UV Raman difference spectrum. In this study, a cooperative double mutant lacking Tyr alpha42, [rHb (Asp beta99 --> Asn, Tyr alpha42 --> Asp)], is used to determine the relative contributions of Tyr alpha42 and the penultimate tyrosines to the R - T UV resonance Raman difference spectrum. The results both directly support the claim that Tyr alpha42 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.
Huang
S
S
Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Peterson
E S
ES
Ho
C
C
Friedman
J M
JM
D000494:Allosteric Regulation; D002263:Carboxyhemoglobin / Q000737:chemistry; D006454:Hemoglobins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D006860:Hydrogen Bonding; D013058:Mass Spectrometry; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D017421:Sequence Analysis; D013059:Spectrum Analysis, Raman; D014443:Tyrosine / Q000737:chemistry; D014466:Ultraviolet Rays
D006454:Hemoglobins; D011994:Recombinant Proteins; D014443:Tyrosine; C100003:deoxyhemoglobin; D002263:Carboxyhemoglobin