PH-INDUCED CONFORMATIONAL-CHANGES OF THE FE2+-N-EPSILON(HIS F8) LINKAGE IN DEOXYHEMOGLOBIN TROUT-IV DETECTED BY THE RAMAN ACTIVE FE2+-N-EPSILON (HIS F8) STRETCHING MODE

To investigate heme-protein coupling via the Fe2+-N(epsilon)(His F8) linkage we have measured the profile of the Raman band due to the Fe2+-N(epsilon)(His F8) stretching mode (nu(Fe-His)) of deoxyHb-trout IV and deoxyHbA at various pH between 6.0 and 9.0. Our data establish that the band of this mode is composed of five different sublines. In deoxyHb-trout IV, three of these sublines were assigned to distinct conformations of the alpha-subunit (OMEGA(alpha-1) = 202 cm-1, OMEGA(alpha-2) = 211 cm-1, OMEGA(alpha-3) = 217 cm-1) and the other two to distinct conformations of the beta-subunit (OMEGA(beta-1) = 223 cm-1 and OMEGA(beta-2) = 228 cm-1). Human deoxyHbA exhibits two alpha-chain sublines at OMEGA(alpha-1) = 203 cm-1, OMEGA(alpha-2) = 212 cm-1 and two beta-chain sublines at OMEGA(beta-1) = 217 cm-1 and OMEGA(beta-2) = 225 cm-1. These results reveal that each subunit exists in different conformations. The intensities of the nu(Fe-His) sublines in deoxyHb-trout IV exhibit a significant pH dependence, whereas the intensities of the corresponding sublines in the deoxyHbA spectrum are independent on pH. This finding suggests that the structural basis of the Bohr effect is different in deoxyHbA and deoxyHb-trout IV. To analyse the pH dependence of the deoxyHb-trout IV sublines we have applied a titration model describing the intensity of each nu(Fe-His) subline as an incoherent superposition of the intensities from sub-sublines with the same frequency but differing intrinsic intensities due to the different protonation states of the respective subunit. The molar fractions of these protonation states are determined by the corresponding Bohr groups (i.e., pK(alpha-1) = pK(alpha-2) = 8.5, pK(beta-1) = 7.5, pK(beta-2) = 7.4) and pH. Hence, the intensities of these sublines reflect the pH dependence of the molar fractions of the involved protonation states. Fitting this model to the pH-dependent line intensities yields a good reproduction of the experimental data. To elucidate the structural basis of the observed results we have employed models proposed by Bangchoroenpaurpong, O., K. T. Schomaker, and P. M. Champion. (1984. J. Am. Chem. Soc. 106:5688-5698) and Friedman, J. M., B. F. Campbell, and R. W. Noble. (1990. Biophys. Chem. 37:43-59) which describe the coupling between the sigma* orbitals of the Fe2+-N(epsilon)(His F8) bond and the pi* orbitals of the pyrrole nitrogens in terms of the tilt angle THETA between its Fe2+-N(epsilon)(His F8)-bond and the heme normal and the azimuthal angle phi between the Fe2+-N(epsilon)(His F8) projection on the heme and the N1-N3 axis. Our results indicate that each subconformation reflected by different frequencies of the nu(Fe-His)-subline is related to different tilt angles THETA, whereas the pH-induced intensity variations of each nu(Fe-His) subline of the deoxyHb trout IV spectrum are caused by changes of the azimuthal angle phi.