SULPHITE REDUCTASE FROM BAKERS YEAST - A HAEMOFLAVOPROTEIN

1. The NADPH-linked sulphite reductase (EC 1.8.1.2) has been purified 300-fold. 2. The stoichiometry for NADPH/S2- is 3:1 and for SO32-/S2- 1:1. 3. Inhibition studies indicate a requirement for thiol groups, flavin and iron for enzyme activity. A CO inhibition which was reversed by light, suggests the participation of a haemprotein. 4. The enzyme is stimulated by riboflavin, FMN or FAD and the Km values are 4.6, 6.3 and 8.3·10-5 M, respectively. 5. The participation of flavin and haemprotein for the reaction is further confirmed by a difference spectrum obtained by adding sulphite to the enzyme. Difference spectra of oxidized versus reduced enzyme are typical of a haemoflavoprotein. Moreover, in a difference spectrum reduced versus reduced plus CO, a Soret peak (416 mμ) and trough at 432 mμ, is similar to that of cytochrome o. 6. During enzyme action, gaseous products were not detected in a mass spectrometer, nor were any free sulphur intermediates found when [35S]sulphite was used. 7. A spectrophotometric method for determining SO32- using 5,5′-dithiobis-(2-nitrobenzoic acid) has been developed. 8. A model for the electron transfer sequence for sulphite reductase is presented. © 1969.