Biophysical Journal 69: 214-227 (1995)
© 1995 the Biophysical Society

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Thermal fluctuations between conformational substates of the Fe(2+)-HisF8 linkage in deoxymyoglobin probed by the Raman active Fe-N epsilon (HisF8) stretching vibration.

FB1-Institut für Experimentelle Physik, Universität Bremen, Germany.

ABSTRACT

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).(ABSTRACT TRUNCATED AT 400 WORDS)

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