Spectral differences between rhodanese catalytic intermediates unrelated to enzyme conformation

Circular dichroism (CD) spectra and UV absorption spectra of two obligatory intermediates in rhodanese catalysis were compared. A broad CD band between 250 and 287 nm increased in a manner stoichiometrically related to the content of enzyme-bound persulfide. Titration of a sample of sulfur-substituted rhodanese (ES) with either cyanide or sulfite gave a stoichiometry that is consistent with one persulfide/molecule of rhodanese (Mr = 33,000). This result agrees with that determined by x-ray crystallography and a method based on quenching of intrinsic fluorescence. Cyanolysis of the persulfide in ES is accompanied by a decrease of UV absorption in the region between 250 and 300 nm. Cyanide titrations followed by the change in absorbance at 263, 272, and 292 nm gave the expected stoichiometry. The magnitude of the difference between the far UV-CD spectra of E and ES found here is smaller than reported previously. This variability suggests that the differences in the secondary structure of these intermediates may not be obligatorily related to the cyanolysis of the persulfide. This view is compatible with recent evidence which suggested that E and ES may be made different by structural relaxation events that occur outside of the catalytic cycle. Furthermore, the methods developed here will be useful in studies on the stability of the catalytic persulfide that has been suggested to be central in the mechanism of several enzymes important in sulfur metabolism.