Studies on yeast sulfite reductase. IV. Structure and steady-state kinetics

Yeast NADPH-sulfite reductase (hydrogen sulfide:NADP+ oxidoreductase, EC 1.8.1.2) is a complex hemoflavoprotein. The Strokes radius was determined to be 80 A by gel filtration and the molecular weight was estimated to be 604,000. The minimal molecular weight calculated from flavin and heme content was 306,000, indicating that this enzyme contains two FAD, two FMN and two hemes per molecule. The enzyme consists of two types of subunit, alpha and beta, having molecular weights of 116,000 and 167,000, respectively. The subunit structure is suggested to be alpha 2 beta 2. The secondary structure, the amino acid composition and the isoelectric point were also investigated. The Km values for sulfite and NADPH were 17 microM and 10 microM, respectively. Sulfite and NADPH affected the reaction velocity to give parallel Lineweaver-Burk plots, indicating the involvement of the 'ping-pong' mechanism in the overall reaction. NADP+ inhibited the reaction competitively with NADPH and noncompetitively with sulfite. Inhibition by sulfide was partially noncompetitive with both substrates but very weak. These results are discussed and compared with sulfite reductase from Escherichia coli.