Circular Dichroism of Cytochrome cc' and Cytochrome c'

Abstract

The circular dichroism (CD)^** of cytochrome cc' from Rhodospirillum rubrum and cytochrome c' from Rhodopseudomonas palustris was measured in the 205-500 mμ region under various conditions where the cytochromes show different types of absorption spectra. At pH 7 the oxidized form of cytochrome cc' exhibits a positive CD extremum at 406mμ in the Soret region, a broad negative CD band from 325 to 360mμ in the o region, and positive extrema between 260 and 285mμ in the aromatic region. The CD spectrum in the far-ultraviolet region exhibits negative extrema at 222 and 207 mμ, which are characteristic of the α-helix. The helical content is calculated to be about 63%. The CD spectra in the Soret, δ, and aromatic region can be changed by the environment concomitant with changes in the absorption spectra. Changes in the CD spectrum in the aromatic region, which are parallel to those in the Soret region, indicate that the CD bands in the aromatic region reflect the environment of the heme groups. Addition of 25% 2-propanol to cytochrome cc', which results in the conversion of the absorp-tion spectrum and the CD spectrum in the Soret, δ, and aromatic regions from the neutral to the alkaline type, causes essentially no change in the CD spectrum in the far-ultraviolet region. In the presence of 2M KCl at pH 12.3, where the absorption spectrum is of the intermediate type, the CD spectrum in the far-ultraviolet region is also the same intermediate type. In the absence of KCI the negative band at 222mμ is greatly diminished at pH 12.3. Treatment with 6M urea for 20 hr at room tem-perature at pH 7 causes considerable destruction of the helical structure but produces only a small change toward the alkaline type in the Soret, δ, and aromatic regions. Only slight changes are found in the whole wavelength range when the sample is examined immediately after the addition of urea. Similar results are obtained on cytochrome c'.
These findings suggest that the change in the absorption spectrum from the neutral to the alkaline type is not necessarily accompanied by a change in the gross helical conformation of the protein moiety which might protect the special environment of the heme.