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 (Hb*CO) 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 Hb*CO 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...

We have measured the absorption spectrum of Ni(II) octaethylporphyrin in CH2Cl2 and in a 50% v/v isopentane/ethyl ether mixture as a function of temperature between 150 and 300 K and 40 and 300 K, respectively. The Soret band can be decomposed into two subbands whose frequencies differ by 220 cm-1. By analogy with resonance Raman results (Jentzen et al. J. Phys. Chem. 1996, 100, 14184−14191 (preceding paper)), we attribute the low-frequency subband to a conformer with a nonplanar macrocycle structure, whereas the high-frequency subband is interpreted as resulting from a planar conformer. The subbands' intensity ratios exhibit a solvent-dependent van't Hoff behavior between 300 and 160 K. Crystallization of CH2Cl2 prevents measurements at lower temperatures. For Ni(II) octaethylporphyrin in the glass-forming isopentane/ethyl ether mixture, the intensity ratio bends over in a region between 150 and 100 K and remains constant below. These data can be fitted by a modified van't Hoff expression which also accounts for the freezing of the above conformers into a nonequilibrium distribution below a distinct temperature T f. The fit yields a freezing temperature of T f = 121 K and a transition region of 52 K. In accordance with the Raman data we found that the nonplanar conformer has the lowest free energy and is therefore dominantly occupied at low temperatures. Furthermore we found that the Soret band's profile is Voigtian with a temperature-dependent Gaussian contribution. The latter results from a bath of low-frequency modes to which the electronic transition into the B state is vibronically coupled. This most likely comprises out-of-plane modes of the porphyrin, in particular those involving the central metal atom, and molecular motions within the liquid environment. At temperatures above the glass transition of the solvent, the amplitudes of these motions increase above the values predicted by a purely harmonic model. This is indicative of strong nonharmonic contributions to their potential energy.

We have measured the VFe-His Raman band of horse heart deoxymyoglobin dissolved in an aqueous solution as a function of temperature between 10 and 300 K. The minimal model to which these data can be fitted in a statistically significant and physically meaningful way comprises four different Lorentzian bands with frequencies at 197, 209, 218, and 226 cm-1, and a Gaussian band at 240 cm-1, which exhibit halfwidths between 10 and 12.5 cm-1. All these parameters were assumed to be independent of temperature. The temperature dependence of the apparent total band shape's frequency is attributed to an intensity redistribution of the subbands at omega 1 = 209 cm-1, omega 2 = 218 cm-1, and omega 3 = 226 cm-1, which are assigned to Fe-N epsilon (HisF8) stretching modes in different conformational substrates of the Fe-HisF8 linkage. They comprise different out-of-plane displacements of the heme iron. The two remaining bands at 197 and 240 cm-1 result from porphyrin modes. Their intensity ratio is nearly temperature independent. The intensity ratio I3/I2 of the vFe-His subbands exhibits a van't Hoff behavior between 150 and 300 K, bending over in a region between 150 and 80 K, and remains constant between 80 and 10 K, whereas I2/I1 shows a maximum at 170 K and approaches a constant value at 80 K. These data can be fitted by a modified van't Hoff expression, which accounts for the freezing into a non-equilibrium distribution of substates below a distinct temperature Tf and also for the linear temperature dependence of the specific heat of proteins. The latter leads to a temperature dependence of the entropic and enthalpic differences between conformational substates. The fits to the intensity ratios of the vFe-His subbands yield a freezing temperature of Tf = 117 K and a transition region of delta T = 55 K. In comparison we have utilized the above thermodynamic model to reanalyze earlier data on the temperature dependence of the ratio Ao/A1 of two subbands underlying the infrared absorption band of the CO stretching vibration in CO-ligated myoglobin (A. Ansari, J. Berendzen, D. Braunstein, B. R. Cowen, H. Frauenfelder, M. K. Kong, I. E. T. Iben, J. Johnson, P. Ormos, T. B. Sauke, R. Scholl, A. Schulte, P. J. Steinbach, R. D. Vittitow, and R. D. Young, 1987, Biophys. Chem. 26:237-335). This yields thermodynamic parameters, in particular the freezing temperature (Tf = 231 K) and the width of the transition region (AT =8 K), which are significantly different from the corresponding parameters obtained from the above vFe-His data, but very close to values describing the transition of protein bound water from a liquid into an amorphous state. These findings and earlier reported data on the temperature dependence exhibited by the Soret absorption bands of various deoxy and carbonmonoxymyoglobins led us to the conclusion that the fluctuations between conformational substates of the heme environment in carbonmonoxymyoglobin are strongly coupled to motions within the hydration shell, whereas the thermal motions between the substates of...

mWe measured the Y~~-~,~ Raman band of horse heart deoxymyoglobin and human deoxyhemoglobin as a function of temperature between 10 and 300 K. A self-consistent spectral analysis of the deoxymyoglobin Raman band reveals that it is underlied by three different sublines with frequencies at f l , = 209 cm-', i12 = 217 cm-', and R, = 225 cm-' and an identical half-width of 13 cm-l. All these parameters were found to be independent of temperature. These sublines were attributed to different conformational substates of the Fe2+-His F8 linkage, which comprise different out-off-plane displacements of the heme iron and tilt angles of the Fe2+-N,(His F8) bond. The intensity ratio 1J12 exhibits a van't Hoff behavior between 150 and 300 K, bends over in a region between 150 and 80 K, and remains constant at lower temperature. In contrast, I2/I1 shows a maximum at 170 K and approaches a constant value at 80 K. These data can be fitted by a modified van't Hoff expression, which accounts for the freezing into nonequilibrium distributions of substrates in a temperature interval AT around a distinct temperature TI and also for the linear temperature dependence of the protein's specific heat. The fits to the above intensity ratios yield a freezing temperature of TI = 117 K and a transition region of AT = 55 K. The v~~-~~~ Raman band of human deoxyhemoglobin was decomposed into seven sublines with frequencies at 195,202,211,218,226,234, and 240 cm-', with half-widths of 12 cm-'. While the low-frequency sublines are strong at GILCH ET AL.300 K, the high-frequency sublines dominate the band at cryogenic temperatures. In comparison, we also investigated the temperature dependence of the near-infrared band I11 at 760 nm. Band I11 of deoxymyoglobin can be decomposed into two subbands which are 165 cm-l apart. The ratio of their absorption cross sections shows a temperature dependence which parallels that of the ratio I 3 / ( I 2 + I,) of the corresponding Raman sublines. Band I11 of deoxyhemoglobin was decomposed into three subbands, the absorption cross sections of which also depend on temperature , similar to what has been observed for the v~~-~~~ subbands. These observations provide strong evidence that the frequency positions of the subbands of band I11 and the v~~-~~~ sublines are governed by the same coordinates. For both proteins investigated, the frequency positions and the half-widths of the band I11 subbands depend significantly on temperature. This is rationalized in terms of an earlier proposed model (Cupane et al., Eur. Biophys. J. 21; 385 1993) which assumes that the corresponding electronic transition is coupled to a bath of low-frequency modes. Our data indicate that these modes are harmonic below 130 K but become anharmonic above this temperature. This onset of anharmonic motions is interpreted as resulting from conformational transitions within the protein which affect the prostethic group via heme-protein coupling.