Structural heterogeneity of the Fe(2+)-N epsilon (HisF8) bond in various hemoglobin and myoglobin derivatives probed by the Raman-active iron histidine stretching mode.

We have examined the Fe(2+)-N epsilon (HisF8) complex in hemoglobin A (HbA) by measuring the band profile of its Raman-active nu Fe-His stretching mode at pH 6.4, 7.0, and 8.0 using the 441-nm line of a HeCd laser. A line shape analysis revealed that the band can be decomposed into five different sublines at omega 1 = 195 cm-1, omega 2 = 203 cm-1, omega 3 = 212 cm-1, omega 4 = 218 cm-1, and omega 5 = 226 cm-1. To identify these to the contributions from the different subunits we have reanalyzed the nu Fe-His band of the HbA hybrids alpha(Fe)2 beta(Co)2 and alpha(Co)2 beta(Fe)2 reported earlier by Rousseau and Friedman (D. Rousseau and J. M. Friedman. 1988. In Biological Application on Raman Spectroscopy. T. G. Spiro, editor, 133-216). Moreover we have reanalyzed other Raman bands from the literature, namely the nu Fe-His band of the isolated hemoglobin subunits alpha SH- and beta SH-HbA, various hemoglobin mutants (i.e., Hb(TyrC7 alpha-->Phe), Hb(TyrC7 alpha-->His), Hb M-Boston and Hb M-Iwate), N-ethylmaleimide-des(Arg141 alpha) hemoglobin (NES-des(Arg141 alpha)HbA) and photolyzed carbonmonoxide hemoglobin (HbCO) measured 25 ps and 10 ns after photolysis. These molecules are known to exist in different quaternary states. All bands can be decomposed into a set of sublines exhibiting frequencies which are nearly identical to those found for deoxyhemoglobin A. Additional sublines were found to contribute to the nu Fe-His band of NES-des(Arg141 alpha) HbA and the HbCO species. The peak frequencies of the bands are determined by the most intensive sublines. Moreover we have measured the nu Fe-His band of deoxyHbA at 10 K in an aqueous solution and in a 80% glycerol/water mixture. Its subline composition at this temperature depends on the solvent and parallels that of more R-like hemoglobin derivatives. We have also measured the optical charge transfer band III of deoxyHbA at room temperature and found, that at least three subbands are required to fit its asymmetric band shape. This corroborates the findings on the nu Fe-His band in that it is indicative of a heterogeneity of the Fe(2+)-N epsilon(HisF8) bond. Finally we measured the nu Fe-His band of horse heart deoxyMb at different temperatures and decomposed it into three different sublines. In accordance with what was obtained for HbA their intensities rather than their frequencies are temperature-dependent. By comparison with VFe-His bands of some Mb mutants (i.e., Mb(His E7.->Gly) and Mb(HisE7__*Met) we suggest that these sublines may be attributed to different conformations of the heme pocket. Our data show, that the V Fe-His band is governed by at least two different coordinates x and y determining its frequency and intensity, respectively. While the former can be assigned to the tilt angle theta between the Fe2+-NJ(HisF8) bond and the heme normal and/or to the displacement delta of the iron from the heme plane, variations in the intensity may be caused by changes of the azimuthal angle phi formed by the projection of the proximal imidazole and the N(l)-Fe2+-N(III) line of the heme. The sublines are therefore interpreted as resulting from different conformational substates of the Fe2+-N(HisFa) complex which differ in terms of x (theta and/or delta). Each of them may further be subdivided in sub-substates with respect to the coordinate y (theta). Quaternary and tertiary transitions of the protein alter the population of these substates thus giving rise to a redistribution of the VFe-HiS sublines which shifts the corresponding peak frequency to higher values.