The rebinding kinetics of CO to myoglobin after flash photolysis is nonexponential in time below approximately 180 K; the kinetics is governed by a distribution of enthalpic barriers. This distribution results from inhomogeneities in the protein conformation, referred to as conformational substates. Hole-burning experiments on the Soret and IR CO-stretch bands test the assumption that an inhomogeneous distribution of conformational substates results in inhomogeneously broadened spectra. CO was slowly photolyzed at different wavelengths in the Soret band at 10 K. Both the Soret band and the CO-stretch band A1, centered at 1,945 cm-1, shift during photolysis, demonstrating that different wavelengths excite different parts of the distributed population.
We have also done kinetic hole-burning experiments by measuring peak shifts in the Soret and A1 bands as the CO molecules rebind. The shifts indicate that the spectral and enthalpic distributions are correlated. In the A1 band, the spectral and enthalpic distributions are highly correlated while in the Soret the correlation is weak. From the peak shifts in the spectral and kinetic hole-burning experiments the inhomogeneous broadening is estimated to be approximately 15% of the total width in the Soret band and approximately 60% in A1. We have previously measured the tilt angle alpha between the bound CO and the heme normal (Ormos, P., D. Braunstein, H. Frauenfelder, M. K. Hong, S.-L. Lin, T. B. Sauke, and R. D. Young. 1988. Proc. Natl. Acad. Sci. USA. 85:8492–8496) and observed a wave number dependence of the tilt angles within the CO-stretch A bands. Thus the spectral and enthalpic distributions of the A bands are coupled to a heterogeneity of the structure.
Todd B. Sauke, P. Ormos, A. Ansari, D. Braunstein, B. R. Cowen, H. Fraunfelder, M. K. Hong, I. E. T. Iben, P. J. Steinbach, and R. D. Young. "Inhomogeneous broadening in spectral bands of carbonmonoxymyoglobin: The connection between spectral and functional heterogeneity" Biophysical Journal (1990): 191-199.