The influence of structural variations in the F- and FG-helix of the beta-subunit modified oxyHb-NES on the heme structure detected by resonance Raman spectroscopy.
The dispersion of the depolarization ratio of two prominent Raman lines (1,375 cm-1 and 1,638 cm-1) of oxyhemoglobin-N-ethyl succinimide have been examined for pH values between pH = 6.0 and 8.5. Both exhibit a significant pH dependence. Calculation of the Raman tensor in terms of a fifth-order time dependent theory provides information about the pH-dependence of parameters reflecting symmetry classified distortions of the prosthetic heme group. To correlate these distortions with the functional properties of the molecule the following protocol was used: 1) An allosteric model suggested by Herzfeld and Stanley (1974) has been applied to O2-binding curves measured at different pH values between 6.5 and 9.0. From this calculation one obtains both, the energy differences between different molecular conformations and the equilibrium constants of oxygen and proton binding. 2) A titration model was formulated relating each conformation of a molecule to a distinct set of distortion parameters of the heme group. 3) The distortion parameters resulting from the analysis of our Raman data were assigned as an effective value due to incoherent superposition of the distortion parameters related to the different titration states. The application of this procedure yields an excellent reproduction of the pH-dependent effective distortion parameters of both Raman lines investigated. It is shown that the protonation of two tertiary effector groups located in the beta-subunits affect the symmetry of the heme in a contrary manner: the protonation of a His-residue (pK = 8.2, probably His(FG4) beta) causes a symmetric position of the proximal imidazole thus lowering the perturbations of the heme core. Further it influences the interaction between amino acid residues of the heme cavity and pyrrole side chains (probably Val(FG5) beta-vinyl (pyrrole 3) thus causing a decrease of the distortions related to the peripheral part of the heme. In contrast, the protonation of Lys (EF6) beta causes a tilt position of the proximal imidazole and an increase of asymmetric perturbations of the heme core, whereas the interaction between the pyrrole side chains and the heme cavity is weakened. Our results are consistent with stereochemical predictions of Moffat (1971) concerning the existence of a H-bond between His(FG4) beta and Cys(F9) beta.
Fachbereich 1, Institut für Physik, Universität Bremen, Federal Republic of Germany.
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Schweitzer-Stenner
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Wedekind
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Dreybrodt
D000328:Adult; D001059:Apoproteins; D006418:Heme; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008968:Molecular Conformation; D010100:Oxygen / Q000378:metabolism; D010108:Oxyhemoglobins; D013059:Spectrum Analysis, Raman; D013388:Succinimides
D001059:Apoproteins; D010108:Oxyhemoglobins; D013388:Succinimides; C060903:oxyhemoglobin-N-ethylsuccinimide; D006418:Heme; D010100:Oxygen