Inhomogeneous broadening in spectral bands of carbonmonoxymyoglobin. The connection between spectral and functional heterogeneity

Abstract

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.

Bacteriorhodopsin photocycle at cryogenic temperatures reveals distributed barriers of conformational substates

“…More direct methods, such as x-ray scattering (7) and Mössbauer spectroscopy (for review, see ref. 1) and spectroscopic and kinetic hole burning (10)(11)(12), have indeed detected such populations of slightly differing conformations.…”

Conformational substates of ferricytochrome c revealed by combined optical absorption and electronic circular dichroism spectroscopy at cryogenic temperature

“…From the spectroscopic point of view, the statistical substates contribute to the rather large spectral Gaussian widths of protein absorption bands (3-5), while taxonomic substates are responsible for their sub-bands structure, like e.g. in the case of the near infrared charge transfer bands of deoxyhemoglobin, deoxymyoglobin and superoxide dismutase (6,7), and of the infrared stretching band of CO bound to myoglobin (2,8,9). Protein conformational substates have substantial functional relevance, since they are responsible for the heterogeneity observed in many dynamical processes like the binding of ligands to the active site; a role of conformational substates in protein folding/unfolding processes has also been suggested (10-…”

Structural factors controlling ligand binding to myoglobin: A kinetic hole-burning study

“…Because both spectroscopic and functional parameters depend on particular structural properties, they are often correlated. For example, if the same structural parameter governs both the position of a spectral line and the enthalpy barrier for recombination, the rebinding reaction is accompanied by a characteristic spectral shift, and ''kinetic hole burning'' (KHB) can be observed (7)(8)(9)11). Frequently, theoretical models exist that relate spectral and structural changes.…”