Studies on pyrocatechase. 3. Circular dichroism and optical rotatory dispersion.

Abstract The circular dichroism of pyrocatechase exhibited several strong positive bands between 250 and 300 mµ, a moderate negative band at 327 mµ, and a weak and broad negative one around 500 mµ, in addition to a strong negative band at 222 mµ, which is characteristic of an α-helix in proteins. In the optical rotatory dispersion curve of the enzyme were discerned a few faint Cotton effects at 250 to 650 mµ and a strong one at 215 to 250 mµ. The negative circular dichroic bands at 327 and 500 mµ and a part of the positive bands between 250 and 300 mµ were associated with the enzyme activity. Upon removal of the bound iron, the bands at 327 and 500 mµ completely disappeared and those between 250 and 300 mµ were partially diminished. Upon incubation with ferrous ion under aerobic conditions these bands were restored to almost the original level. Therefore, the iron bound to the enzyme may be responsible for the circular dichroic bands as well as the enzyme activity. The disappearance of the bands above 300 mµ also occurred on addition of reducing agents, an indication that the iron in the enzyme is in the trivalent state, which is consistent with the previous results. In the presence of the substrate, catechol, under anerobic conditions, the magnitude and the position of the circular dichroic bands related to the enzyme activity changed, indicating the alteration of the state of ligands around the iron on binding of the substrate.