CONFORMATIONAL SUBSTATES OF THE FE2-HIS F8 LINKAGE IN DEOXYMYOGLOBIN AND HEMOGLOBIN PROBED IN PARALLEL BY THE RAMAN BAND OF THE FE-HIS STRETCHING VIBRATION AND THE NEAR-INFRARED ABSORPTION-BAND-III()

We measured the nu(Fe-His) Raman band of horse heart deoxymyoglobin an d human deoxyhemoglobin as a function of temperature between 10 and 30 0 K. A self-consistent spectral analysis of the deoxymyoglobin Raman b and reveals that it is underlied by three different sublines with freq uencies at Ohm(1) = 209 cm(-1), Ohm(2) = 217 cm(-1), and Ohm(3) = 225 cm(-1) and an identical half-width of 13 cm(-1). All these parameters were found to be independent of temperature. These sublines were attri buted to different conformational substates of the Fe2+-His F8 linkage , which comprise different out-off-plane displacements of the heme iro n and tilt angles of the Fe2+-N-epsilon(His F8) bond. The intensity ra tio I-3/I-2 exhibits a van't Hoff behavior between 150 and 300 K, bend s over in a region between 150 and 80 K, and remains constant at lower temperature. In contrast, I-2/I-1 shows a maximum at 170 K and approa ches a constant value at 80 K. These data can be fitted by a modified van't Hoff expression, which accounts for the freezing into nonequilib rium distributions of substrates in a temperature interval Delta T aro und a distinct temperature T-f and also for the linear temperature dep endence of the protein's specific heat. The fits to the above intensit y ratios yield a freezing temperature of T-f = 117 K and a transition region of Delta T = 55 K. The nu(Fe-His) Raman band of human deoxyhemo globin was decomposed into seven sublines with frequencies at 195, 202 , 211, 218, 226, 234, and 240 cm(-1), with half-widths of 12 cm(-1). W hile the low-frequency sublines are strong at 300 K, the high-frequenc y sublines dominate the band at cryogenic temperatures. In comparison, we also investigated the temperature dependence of the near-infrared band III at 760 nm. Band III of deoxymyoglobin can be decomposed into two subbands which are 165 cm(-1) apart. The ratio of their absorption cross sections shows a temperature dependence which parallels that of the ratio I-3/(I-2 + I-1) of the corresponding Raman sublines. Band I II of deoxyhemoglobin was decomposed into three subbands, the absorpti on cross sections of which also depend on temperature, similar to what has been observed for the nu(Fe-His) subbands. These observations pro vide strong evidence that the frequency positions of the subbands of b and III and the nu(Fe-His) sublines are governed by the same coordinat es. For both proteins investigated, the frequency positions and the ha lf-widths of the band III subbands depend significantly on temperature . This is rationalized in terms of an earlier proposed model (Cupane e t al., Fur. Biophys. J. 21; 385 1993) which assumes that the correspon ding electronic transition is coupled to a bath of low-frequency modes . Our data indicate that these modes are harmonic below 130 K but beco me anharmonic above this temperature. This onset of anharmonic motions is interpreted as resulting from conformational transitions within th e protein which affect the prostethic group via heme-protein coupling. (C) 1996 John Wiley & Sons, Inc.