The Flavin Reductase Activity of the Flavoprotein Component of Sulfite Reductase from Escherichia coli

Sulfite reductase (SiR) from Escherichia coli has a α8β4 subunit structure, where α8 is a flavoprotein (SiR-FP) containing both FAD and FMN as prosthetic groups. It also exhibits a NADPH:flavin oxidoreductase activity with exogenous riboflavin, FMN, and FAD serving as substrates. The flavin reductase activity may function during activation of ribonucleotide reductase or during ferrisiderophore reduction. A plasmid containing cysJ gene, coding for the α subunit, overexpresses flavin reductase activity by 100-fold, showing that α is the site of free flavin reduction. The overproducer allows a fast and simple preparation of large amounts of the flavoprotein. Kinetic studies of its flavin reductase activity demonstrates a ping-pong bisubstrate-biproduct reaction mechanism. NADP inhibition studies show that both substrates, NADPH and free flavins, bind to the same site. While the FAD cofactor mediates the electron transfer between NADPH and free flavins, the FMN cofactor is not essential since a FMN-depleted SiR-FP retains a large proportion of activity. In contradiction with previous reports, SiR-FP is found to contain 1.6-1.7 flavin per α subunit. This result, together with the sequence homology between SiR-FP and NADPH-cytochrome P-450 reductase, suggests a new model for the structure of the protein with one FMN and one FAD prosthetic group per α subunit.