The second derivative electronic absorption spectrum of cytochrome c oxidase in the Soret region

The electronic absorption spectrum of solubilized beef heart cytochrome c o xidase was analyzed in the region to identify the origin of doublet feature s appearing in the second derivative spectrum associated with ferrocytochro me a. This doublet, centered near 22,600 cm(-1), was observed in the direct absorption spectrum of the a(2+)a(3)(3+).HCOO- form of the enzyme at cryog enic temperatures. Since evidence for this doublet at room temperature is o btained only on the basis of the second derivative spectrum, a novel mathem atical approach was developed to analyze the resolving power of second deri vative spectroscopy as a function of parameterization of spectral data. Wit hin the mathematical limits defined for resolving spectral features, it was demonstrated that the integrated intensity of the doublet feature near 450 nm associated with ferrocytochrome a is independent of the ligand and oxid ation state of cytochrome a,. Furthermore, the doublet features, also obser ved in cytochrome c oxidase from Paracoccus denitrificans, were similarly a ssociated with the heme A component and were correspondingly independent of the ligand and oxidation state of the heme A, chromophore. The doublet fea tures 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 Sor et transition.