The flavoprotein component (SiR-FP) of the sulfite reductase from Escherichia coli is an octamer containing one FAD and one FMN as cofactors per polypeptide chain. We have constructed an expression vector containing the DNA fragment encoding for the FMN-binding domain of SiR-FP. The overexpressed protein (SiR-FP23) was purified as a partially flavin-depleted polymer. It could incorporate FMN exclusively upon flavin reconstitution to reach a maximum flavin content of 1.2 per polypeptide chain. Moreover, the protein could stabilize a neutral air-stable semiquinone radical over a wide range of pHs. During photoreduction, the flavin radical accumulated first, followed by the fully reduced state. The redox potentials, determined at room temperature [E'1 (FMNH./FMN) = -130 +/- 10 mV and E'2 (FMNH2/FMNH.) = -335 +/- 10 mV], were very close to those previously reported for Salmonella typhimurium SiR-FP [Ostrowski, J., Barber, M. J., Rueger, D. C., Miller, B. E., Siegel, L. M., & Kredich, N. M. (1989) J. Biol. Chem. 264, 15796-15808]. Both the radical and fully reduced forms of SiR-FP23 were able to transfer their electrons to cytochrome c quantitatively. Altogether, the results presented herein demonstrate that the N-terminal end of E. coli SiR-FP forms the FMN-binding domain. It folds independently, thus retaining the chemical properties of the bound FMN, and provides a good model of the FAD-depleted form of native SiR-FP. Moreover, the FMN prosthetic group in SiR-FP23 and native SiR-FP is compared to that of cytochrome P450 reductase and bacterial cytochrome P450, which also contain one FAD and one FMN per polypeptide chain.