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

Abstract

Sulfite reductase (SiR) from Escherichia coli has a alpha 8 beta 4 subunit structure, where alpha 8 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 alpha subunit, overexpresses flavin reductase activity by 100-fold, showing that alpha 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 alpha 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 alpha subunit.

NADPH‐sulfite reductase flavoprotein from Escherichia coli: Contribution to the flavin content and subunit interaction

“…SiR-FP was purified from the ~ overproducing strain as previously described [7]. Protein concentration was estimated by the method of Bradford using bovine serum albumin as a standard [11].…”

“…NADPH-dependent reactions were carried out as previously described [4,7] with 0.25 mM NADPH, an electron acceptor and an appropriate amount of protein. Electron acceptors were present at the following concentrations: 0.1 mM riboflavin, 0.3 mM ferricyanide, 0.2 mM AcPyADP + and 0.1 mM cytochrome c. Absorbance changes were followed using a Kontron Uvikon 930 spectrophotometer at 340 nm for riboflavin and ferricyanide; at 363 nm for AcPyADP ÷ and at 550 nm for cytochrome c. The following extinction coefficients were used: NADPH, ~340 ----6.22 mM -1 -cm-l; AcPyADP +, e363 = 5.6 mM -l .cm-~; reduced cytochrome c, e550 = 22 mM -~.…”

The FNR-like domain of the Escherichia coli sulfite reductase flavoprotein component: crystallization and preliminary X-ray analysis

“…It has been shown that the two SiR-FP flavinic domains can be separated by proteolytic cleavage between Ser217 and Thr220 (Eschenbrenner et al, 1995b). From the properties of the resulting fragments it can be concluded that the N-terminal domain (SiR-FP23) is responsible for the polymerization of the a-chains (Eschenbrenner et al, 1995b) and that the C-terminal monomeric domain (SiR-FP43) is able to catalyze efficient NADPH-dependent reductions of flee flavins (Eschenbrenner et al, 1995a).…”

“…From the properties of the resulting fragments it can be concluded that the N-terminal domain (SiR-FP23) is responsible for the polymerization of the a-chains (Eschenbrenner et al, 1995b) and that the C-terminal monomeric domain (SiR-FP43) is able to catalyze efficient NADPH-dependent reductions of flee flavins (Eschenbrenner et al, 1995a). SiR-FP43 belongs to the ferredoxin NADPreductase (FNR) structural family which includes FNR's, phthalate dioxygenase reductase (PDR) (Correll et al, 1992), neutrophil NADPH oxidase and flavin reductases.…”

“…SiR-FP also has diaphorase activity and is able to transfer electrons either from FMN site to other acceptors, such as cytochrome c or ferricyanide, or directly from the FAD to AcPyADP + or flee exogenous flavin (Eschenbrenner et al, 1995b).…”