The Second Derivative Electronic Absorption Spectrum of Cytochrome c Oxidase in the Soret Region

AbstractThe electronic absorption spectrum of solubilized beef heart cytochrome c oxidase was analyzed in the 400–500nm region to identify the origin of doublet features appearing in the second derivative spectrum associated with ferrocytochrome a. This doublet, centered near 22,600cm−1, was observed in the direct absorption spectrum of the a2+a33+·HCOO− form of the enzyme at cryogenic temperatures. Since evidence for this doublet at room temperature is obtained only on the basis of the second derivative spectrum, a novel mathematical approach was developed to analyze the resolving power of second derivative spectroscopy as a function of parameterization of spectral data. Within the mathematical limits defined for resolving spectral features, it was demonstrated that the integrated intensity of the doublet feature near 450nm associated with ferrocytochrome a is independent of the ligand and oxidation state of cytochrome a3. Furthermore, the doublet features, also observed in cytochrome c oxidase from Paracoccus denitrificans, were similarly associated with the heme A component and were correspondingly independent of the ligand and oxidation state of the heme A3 chromophore. The doublet features are attributed to lifting of the degeneracy of the x and y polarized components of the B state of the heme A chromophore associated with the Soret transition