Biophysical Journal 64: 1194-1209 (1993)
© 1993 the Biophysical Society

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Raman dispersion spectroscopy probes heme distortions in deoxyHb-trout IV involved in its T-state Bohr effect

Institute of Experimental Physics, University of Bremen, 2800 Bremen 33, Germany

ABSTRACT

The depolarization ratios of heme protein Raman lines arising from vibrations of the heme group exhibit significant dependence on the excitation wavelength. From the analysis of this depolarization ratio dispersion, one obtains information about symmetry-lowering distortions Q of the heme group that can be classified in terms of the symmetry races = A_1g, B_1g, B_2g, and A_2g in D_4h symmetry. The heme-protein interaction can be changed by the protonation of distinct amino acid side chains (i.e., for instance the Bohr groups in hemoglobin derivates), which gives rise to specific static heme distortions for each protonation state. From the Raman dispersion data, it is possible to obtain parameters by fitting to a theoretical expression of the Raman tensor, which provide information on these static distortions and also about the pK values of the involved titrable side chains. We have applied this method to the ₄ (1,355 cm^-1) and ₁₀ (1,620 cm^-1) lines of deoxygenated hemoglobin of the fourth component of trout and have measured their depolarization ratio dispersion as a function of pH between 6 and 9. From the pH dependence of the thus derived parameters, we obtain pK values identical to those of the Bohr groups, which were earlier derived from the corresponding O₂-binding isotherms. These are pK₁ = pK₂ = 8.5 for the and pK_ß1 = 7.5, pK_ß2 = 7.4 for the ß chains. We also obtain the specific distortion parameters for each protonation state. As shown in earlier studies, the ₄ mode mainly probes distortions from interactions between the proximal histidine and atoms of the heme core (i.e., the nitrogens and the C atoms of the pyrroles). Group theoretical argumentation allows us to relate specific changes of the imidazole geometry as determined by its tilt and azimuthal angle and the iron-out-of-plane displacement to distinct variations of the normal distortions Q derived from the Raman dispersion data. Thus, we found that the pH dependence of the heme distortions Q^A1g (totally symmetric) and Q^B1g (asymmetric) is caused by variations of the azimuthal rather than the tilt angle of the Fe-His (F8) bond. In contrast to this, the ₁₀ line mainly monitors changes resulting from the interaction between peripheral substituents of the porphyrin macrocycle (vinyl). From the pH dependence of the parameters, it is possible to separately identify distortions Q affecting the hemes in the and ß chains, respectively. From this, we find that in the subunit structural changes induced on protonation of the corresponding Bohr groups are mainly transferred via the Fe—N bond and give rise to changes in the azimuthal angle. In the ß subunit, however, in addition, structural changes of the heme pocket arise, which most probably result from protonation of the imidazole of the COOH-terminal His (HC3 ß). This rearranges the net of H bonds between His HC3 ß, Ser (F9 ß), and Glu (F7 ß).