NADPH-SULFITE REDUCTASE FROM ESCHERICHIA-COLI - A FLAVIN REDUCTASE PARTICIPATING IN THE GENERATION OF THE FREE-RADICAL OF RIBONUCLEOTIDE REDUCTASE

Protein R2, the small subunit of ribonucleotide reductase of Escherich ia coli, contains an essential free radical localized to tyrosine 122 of its polypeptide chain. When this radical is scavenged by hydroxyure a, the enzyme is transformed into an inactive form, metR2. E. coli con tains a NAD(P)H:flavin oxidoreductase, named Fre, absolutely required for the regeneration of the radical and the activation of metR2 into R 2. Consequently, an E. coli mutant strain lacking an active fre gene i s more sensitive to hydroxyurea during growth, demonstrating the physi ological protective function of Fre from the loss of the radical. Howe ver, this gene is not essential, and we found that E. coli contains a second tyrosyl radical generating activity, also residing in a flavin reductase. The enzyme has been purified 200-fold to homogeneity and fo und to be identical to sulfite reductase. Pure sulfite reductase has t he ability to catalyze the reduction of free riboflavin, FMN, or FAD b y NADPH and thus, as Fre, to transfer electrons to the iron center of metR2, a key step during the activation reaction.