Conformational Changes in FmetHbS Probed with UV Resonance Raman and Fluorescence Spectroscopic Methods

Abstract

The tertiary and quaternary structure of HbS has been investigated using UV resonance Raman (UVRR) and fluorescence spectroscopic methods. This well-characterized Hb mutant (β6 Glu → Val), which forms polymers under deoxygenating conditions, was studied in the tetrameric form as the fluoromet derivative and compared with HbA under the same conditions. The excitation wavelengths employed in this study preferentially probe the Tyr and Trp residues in the protein. Comparison of UVRR and fluorescence difference spectra generated between the T and R states of FmetHbA and FmetHbS are indicative that the α1β2 intersubunit contacts, monitored through the β37 Trp and the α42 Tyr residues, are essentially the same for the two hemoglobins. (The abbreviations used are the following: Hb, hemoglobin; FmetHbA, FmetHbS, the fluoromet derivatives of hemoglobin A and hemoglobin S; UVRR, ultraviolet resonance Raman, IHP, inositol hexaphosphate.) The tertiary conformation of FmetHbS, however, is perturbed relative to that of FmetHbA in both the T and R quaternary states, as shown by an overall increase in the intensity of the Trp modes in the UVRR spectra and a decrease in the fluorescence intensity. These spectral differences are attributed to the β15 Trp residue, because of the mutation at the β6 position. The combined spectroscopic studies provide evidence for an altered tertiary structure in HbS where the A-helix is displaced toward the E-helix as monitored by the strength of the β15 Trp···β72 Ser H-bond.