Spectral splitting in the alpha (Q(0,0)) absorption band of ferrous cytochrome c and other heme proteins

The alpha or Q(0,0) absorption band of horse iron(II) cytochrome c splits and shifts to the blue as temperature decreases over the temperature range of 290-10 K. At room temperature, its maximum is at 18 150 cm(-1) and the spectral width is 273 cm(-1), whereas at 10 K, the two bands of the Q(0,0) transition occur at 18 364 and 18 253 cm(-1) and the width of the lowest-energy band is 96 cm(-1). Temperature dependent splitting also occurs for zinc cytochrome c, a derivative in which Fe has been replaced by Zn; at 10 K, the peaks in the Q(0,0) band region occur at 17 106 and 16 996 cm(-1). The peak positions are independent of the cryosolvent (aqueous ethylene glycol or glycerol mixtures). The splitting of the Q(0.0) band seen in the protein (similar to 110 cm(-1) for iron and zinc cytochrome c) is comparable to the crystal field splitting observed for metalloporphyrins in mixed crystals. In contrast, the Q(0,0) band of zinc coproporphyrin III in a glassy solvent (dimethylformamide/ethylene glycol) or in poly(vinyl chloride) shows a blue shift with temperature decrease but no evidence of Q(0,0) splitting. Available spectral data show that the Q(0,0) band is composed of two nearly degenerate electronic transitions and the split is due to the asymmetry in the heme pocket of the protein that arises from the surrounding polypeptide chain. This asymmetry results in the stabilization of one form of the excited slate over the other, according to a Jahn-Teller mechanism.