Optical Rotatory Dispersion of Some c' and cc' Cytochromes

Abstract

Abstract The optical rotatory dispersion behavior of the oxidized, reduced, and carbon monoxide-reduced forms of cytochrome c' from Rhodopseudomonas palustris and cytochrome cc' from Chromatium strain D, Rhodospirillum rubrum, and Pseudomonas dinitrificans, was studied in the range from 600 to 185 nm. These cytochromes exhibited an apparent positive Cotton effect in the Soret region characteristically different from other c-types cytochromes. The experimental ORD curves were resolved into individual Cotton effects plus the protein backbone rotations which were then also transformed into circular dichroic spectra. The results showed that in all three states, the anomalous rotations occurring within the Soret band were composed of at least two or three positive Cotton effects of relatively narrow band width and large rotational strength; a negative Cotton effect, evidently composed of multiple transitions, was resolved in the 300- to 330-nm region. For both the reduced and the carbon monoxide-reduced forms, the main Cotton effects were shifted towards longer wave length and in the carbon monoxide-reduced form, with the exception of R. rubrum, these Cotton effects occurred at positions close to the absorption maximum of the cytochrome and possessed very narrow band width. The net rotational strength (sum of Cotton effects I, II, and III) was significantly decreased for R. palustris cytochrome c' when reduced and for Chromatium and R. rubrum cytochrome cc' when reacted with carbon monoxide. The Shechter-Blout parameters (A'193 nm and A'225 nm) were found, in general, to be dependent upon not only the oxidation state of the cytochromes but also the ligand coordinated with the heme iron. These observations suggested that changes in the heme chromophore or chromophores also resulted in alterations of the heme environment. No evidence of heme-heme interactions was evident for the diheme cytochromes from Chromatium, R. rubrum and P. dinitrificans in contrast to a diheme cytochrome from Chromatium, cytochrome c552. Distinct differences were noted in the optical rotatory dispersion spectra and in the resolved Cotton effects between cytochrome cc' from Chromatium and R. rubrum, and between the monoheme (R. palustris) and the diheme cytochromes.