RAMAN DISPERSION SPECTROSCOPY PROBES HEME DISTORTIONS IN DEOXYHB-TROUT IV INVOLVED IN ITS T-STATE BOHR EFFECT

The depolarization ratios of heme protein Raman lines arising from vib rations of the heme group exhibit significant dependence on the excita tion wavelength. From the analysis of this depolarization ratio disper sion, one obtains information about symmetry-lowering distortions delt aQ(GAMMA) of the heme group that can be classified in terms of the sym metry races GAMMA = A1g, B1g, B2g, and A2g in D4h symmetry. The heme-p rotein interaction can be changed by the protonation of distinct amino acid side chains (i.e., for instance the Bohr groups in hemoglobin de rivates), which gives rise to specific static heme distortions for eac h 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 als o about the pK values of the involved titrable side chains. We have ap plied this method to the nu4 (1,355 cm-1) and nu10 (1,620 cm-1) lines of deoxygenated hemoglobin of the fourth component of trout and have m easured their depolarization ratio dispersion as a function of pH betw een 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 ea rlier derived from the corresponding O2-binding isotherms. These are p K(alpha1) = pK(alpha2) = 8.5 for the alpha and pK(beta1) = 7.5, pK(bet a2) = 7.4 for the beta chains. We also obtain the specific distortion parameters for each protonation state. As shown in earlier studies, th e nu4 mode mainly probes distortions from interactions between the pro ximal histidine and atoms of the heme core (i.e., the nitrogens and th e C(alpha) atoms of the pyrroles). Group theoretical argumentation all ows us to relate specific changes of the imidazole geometry as determi ned by its tilt and azimuthal angle and the iron-out-of-plane displace ment to distinct variations of the normal distortions deltaQ(GAMMA) de rived from the Raman dispersion data. Thus, we found that the pH depen dence of the heme distortions deltaQ(A1g) (totally symmetric) and delt aQ(B1g) (asymmetric) is caused by variations of the azimuthal rather t han the tilt angle of the Fe-His (F8) bond. In contrast to this, the n u10 line mainly monitors changes resulting from the interaction betwee n peripheral substituents of the porphyrin macrocycle (vinyl). From th e pH dependence of the parameters, it is possible to separately identi fy distortions deltaQ(GAMMA) affecting the hemes in the alpha and beta chains, respectively. From this, we find that in the a subunit struct ural changes induced on protonation of the corresponding Bohr groups a re mainly transferred via the Fe - N(epsilon) bond and give rise to ch anges in the azimuthal angle. In the beta subunit, however, in additio n, structural changes of the heme pocket arise, which most probably re sult from protonation of the imidazole of the COOH-terminal His (HC3 b eta). This rearranges the net of H bonds between His HC3 beta, Ser (F9 beta), and Glu (F7 beta).