Studies on yeast sulfite reductase: III. Further characterization

Abstract

1. 1. Acid ammonium sulfate treatment of purified yeast NADPH-sulfite reductase caused selective detachment of FMN from the protein, leaving FAD still attached to the precipitated enzyme. The NADPH-linked activities of the treated enzyme could be partly restored by the addition of FMN or FAD. 2. 2. The NADPH-linked activities, but not the reduced methyl viologen (MVH)-sulfite reductase activity, of the enzyme were inactivated on exposure to low ionic strength. This inactivation was accompanied by a decrease in sedimentation coefficient for the enzyme protein. The NADPH-sulfite reductase activity thus inactivated could be restored, though to very limited extents, by the addition of a protein fraction from yeast mutants incapable of reducing sulfite. 3. 3. The presence in yeast crude extracts of at least two types of NADPH-cytochrome c reductase activity was demonstrated; one type of activity was associated with the sulfite reductase and sensitive to low salt concentrations, whereas the other was stable to low ionic strength. The sensitive reductase activity was absent in all the mutant strains which are genetically blocked in the sulfite reduction step. 4. 4. Heat treatment of the purified enzyme decreased the heights of absorption peaks at 587 and 386 mμ, and the loss of the MVH-sulfite reductase activity paralleled the decrease in the intensity of the 587-mμ peak. 5. 5. The results of ultraviolet irradiation of the enzyme provided evidence that the 587-mμ chromophore also absorbs in the 386-mμ region. 6. 6. The enzyme possessed two different fluorescent species; one seemed to be flavin(s) and the other was probably the 587-mμ chromophore. 7. 7. The ESR spectrum of the reduced enzyme, but not of the oxidized enzyme, showed a sharp signal at g = 2.