The Heme Chromophore in the Ultraviolet

Abstract

Absorption, optical rotatory dispersion, and circular dichroism data are presented for the oxidized and reduced states of the heme undecapeptide of cytochrome c. The absence of aromatic residues, disulfides, and ionizable sulfhydryl groups allows unambiguous determination of the heme absorptions to 240 mµ. As the electronic transitions within the heme are made optically active by the covalently attached peptide, it is possible to detect bands not readily resolved from absorption data. An absorption maximum is seen at 277 mµ which is labeled the e hemochromogen band. In the presence of imidazole, optically active bands are found in the ferroheme undecapeptide with extrema at 263 mµ, 278 mµ, 317 mµ, and 353 mµ. The ferriheme undecapeptide exhibits a positive circular dichroism band at 253 mµ and a broad negative extremum in the 320- to 360-mµ wavelength range. These bands, including those which are found in the wavelength range normally reserved to the aromatic amino acids, are due to the heme chromophore. At shorter wavelengths it is shown that the absorption at 190 mµ cannot be adequately accounted for by the peptide moiety and the carboxyls of the propionic acid heme substituents, that is, 25 to 50% of the absorption at 190 mµ is due to heme, the coordinated imidazole, and the thiol ether groups. The circular dichroism in the peptide region is dependent on the oxidative state and indicates changes directly involving the heme moiety either electronically or sterically. The bands observed in the heme peptides are briefly compared to qualitatively similar bands in cytochrome c, myoglobin, and hemoglobin. The distinctive feature in the circular dichroism of the heme proteins is the presence of localized bands which correspond to vibrational bands present in the aromatic amino acids. The vibronic bands should allow assessment of environmental changes in the corresponding amino acids.