A spectrophotometric and fluorimetric study of alkaline transitions of Euglena cytochrome c 552.
The behavior of the photosynthetic cytochrome c552 upon titration with alkali depends on the ionic composition of the medium. In water the disappearance of the 695-nm band, indicating the displacement of the methionine ligand, as well as a remarkable tryptophan fluorescense enhancement, follow a single proton titration curve with pK of 10.0 and n=1.0. The product is a low spin type protein. In salt-containing media two successive steps are observed: in the first one, completed at about pH 10.3, a high-spin form of cytochrome c 552 is obtained and relatively small fluorescence enhancement is detected. In the second step, more profound fluorometric changes occur, while the material reverts to its low-spin form. Addition of salts to an alkaline solution of cytochrome c 552 in water results in the formation of a 600-nm high-spin band with a concomitant quenching of tryptophan fluorescence. The results imply that at high pH unfolding of the molecule is evident only when the low-spin product is obtained. In the high-spin alkaline form, the methionine ligand is probably displaced from iron coordination by hydroxyl ions, while in the low-spin alkaline form methionine may be replaced by a lysyl residue of the cytochrome c 552 protein. The results imply that the lysyl residue is available for coordination in salt solutions at a higher pH than in water.
A spectrophotometric and fluorimetric study of alkaline transitions of Euglena cytochrome c 552. The behavior of the photosynthetic cytochrome c552 upon titration with alkali depends on the ionic composition of the medium. In water the disappearance of the 695-nm band, indicating the displacement of the methionine ligand, as well as a remarkable tryptophan fluorescense enhancement, follow a single proton titration curve with pK of 10.0 and n=1.0. The product is a low spin type protein. In salt-containing media two successive steps are observed: in the first one, completed at about pH 10.3, a high-spin form of cytochrome c 552 is obtained and relatively small fluorescence enhancement is detected. In the second step, more profound fluorometric changes occur, while the material reverts to its low-spin form. Addition of salts to an alkaline solution of cytochrome c 552 in water results in the formation of a 600-nm high-spin band with a concomitant quenching of tryptophan fluorescence. The results imply that at high pH unfolding of the molecule is evident only when the low-spin product is obtained. In the high-spin alkaline form, the methionine ligand is probably displaced from iron coordination by hydroxyl ions, while in the low-spin alkaline form methionine may be replaced by a lysyl residue of the cytochrome c 552 protein. The results imply that the lysyl residue is available for coordination in salt solutions at a higher pH than in water.