Electronic Spectrum of Single Crystals of Ferricytochrome-c

The absorption spectrum of crystalline horse-heart ferricytochrome-c was investigated. Measurements were made on single crystals at room temperature from 3700 to 7600 Å using a microspectrophotometer. Molar extinction coefficients for light parallel and perpendicular to the fourfold c crystallographic axis are given. The Soret band (4100 Å) and main visible band (5300 Å) were found to have the same polarization, while the weaker band at 5650 Å is polarized differently. These transitions are discussed in terms of the molecular orbital theory for simple metalloporphyrin complexes. Assuming the Soret and main visible bands correspond to transitions to an Eu state, the angle that the normal to the porphyrin plane makes with the c axis is calculated from the polarization ratios to be 72°. Using this angle the isotropic extinction coefficients calculated from the crystal data agree well with the extinction coefficients for these bands in solution. The weak band at 6950 Å is polarized out of the porphyrin plane and is tentatively ascribed to the promotion of an electron from the porphyrin a2u(π) orbital to the iron a1g(dz2) orbital. The intensity of this band has been shown to be dependent on the conformation of the polypeptide. The proposed mechanism for this transition indicates two different ligand-heme configurations in ferricytochrome-c which are consequent upon the two ``conformational isomers'' postulated by Schejter and George. It is concluded that hemoproteins provide a near perfect example of an oriented gas of chromophores.