In all heme proteins the structure and properties of the heme crucially controls the functioning of the transport protein. For no other prosthetic group is there such an enticing relationship between the structure, spectra and function of the group, nor such a well-defined link between the properties of the active center and the function of the protein. Cytochrome c's (cyt c) are small hemoproteins involved in electron transfer in all cells that have respiration. By definition, the hemes are covalently attached by condensation of vinyl groups in c type cytochromes. The structure of many cyt c's are known, allowing for structure/function correlations, and these proteins have long served as models to study the general question of electron transfer in hemoproteins. In this work we are addressing a 50 year old unsolved problem regarding cyt c. Keilin in 1949 reported that the absorption spectra of some Fe(II) heme proteins sharpen and the Q0,0 or ?-band splits as temp erature lo wers. In spite of numerous studies on heme proteins, the origin of the temperature dependence of the spectrum and its significance, if any, to electron transfer functions has not been generally recognized by people working in the area of hemoproteins. In the literature, various models have been proposed for origin of the split. In one model, there are two populations of proteins, one absorbing at lower and the other at higher wavelength. This interpretation is especially popular, since other experiments indicate that proteins do have subconformations. In another model, there are two electronic transitions. These correspond to levels that are degenerate, but in an asymmetric environment (such as the heme pocket), one level is stabilized relative to the other. All molecules within the sample would have the same absorption spectrum. By using fluorescence anisotropy we are discerning which of these various models is the correct one. These experiments are being performed on the time-correlated instrument at the RLBL.
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