Spectroscopic studies of structural changes in hemoglobins

Abstract

In this thesis, we investigate several hemoglobins using a variety of biophysical and biochemical techniques.;Ascaris hemoalobin. The Ascaris hemoglobin has a very low spontaneous dissociation rate for the dioxygen ligand. UV Raman spectra of deoxy, CO and oxy forms of domain one Ascaris hemoglobin (D1) were acquired and compared. The data show that the binding of CO or O{dollar}\sb 2{dollar} to this hemoglobin induces the same conformational change. The major spectral differences between CO and oxy D1 are shown in the tyrosine bands which are sensitive to hydrogen bonding. These results provide the direct evidence that tyrosine B10 is hydrogen bonded to the bound oxygen, resulting in a low oxygen dissociation rate.;Hb C. The structure of oxyhemoglobin C, a hemoglobin A mutant (Glu {dollar}\rm\beta6\to Lys{dollar}) was probed by fluorescence and Raman spectroscopy. A significant difference in the fluorescence intensity of Oxy Hb C and oxyHb A is observed. The UV Raman data point to a altered environment of Trp {dollar}\beta{dollar}15 in Hb C as the source of difference in the fluorescence intensity. Together the results suggest that the hydrogen bond between Trp {dollar}\beta{dollar}15 and Ser {dollar}\beta{dollar}72 is weaker in Hb C, resulting in a displacement of the A helix from the E helix.;Hb A. Chapter Five explores the structural and functional role of the penultimate tyrosine residues in hemoglobin A. The UV Raman results obtained from a double mutant Hb A (r Hb, {dollar}\beta{dollar}99 Asp {dollar}\to{dollar} Asn; {dollar}\alpha{dollar}42 Tyr {dollar}\to{dollar} Asp) support the claim that Tyr {dollar}\alpha{dollar}42 is the proton acceptor when hydrogen-bonded to Asp {dollar}\beta{dollar}99 in the T state. This hydrogen bonding arrangement suggests that this tyrosine residue is located in an extremely hydrophobic environment. The elimination of UV Raman signals from this pair of residues makes it possible to monitor other tyrosines during quaternary structural change. The results indicate a T-state strengthening of the hydrogen bond between the penultimate tyrosines and their hydrogen bonding partners at the FG corners. The change in the hydrogen bonding strength may result in the modulation of heme environments and ligand binding behavior.