FLAVIN MONONUCLEOTIDE-BINDING DOMAIN OF THE FLAVOPROTEIN COMPONENT OFTHE SULFITE REDUCTASE FROM ESCHERICHIA-COLI

The flavoprotein component (SiR-FP) of the sulfite reductase from Esch erichia coli is an octamer containing one FAD and one FMN as cofactors per polypeptide chain. We have constructed an expression vector conta ining the DNA fragment encoding for the FMN-binding domain of SiR-FP. The overexpressed protein (SiR-FP23) was purified as a partially flavi n-depleted polymer. It could incorporate FMN exclusively upon flavin r econstitution to reach a maximum flavin content of 1.2 per polypeptide chain. Moreover, the protein could stabilize a neutral air-stable sem iquinone 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 t he radical and fully reduced forms of SiR-FP23 were able to transfer t heir electrons to cytochrome c quantitatively. Altogether, the results presented herein demonstrate that the N-terminal end of E. coli SiR-F P forms the FT;IN-binding domain: It folds independently, thus retaini ng the chemical properties of the bound FMN, and provides a good model of the FAD-depleted form of native SiR-FP. Moreover, the FMN prosthet ic group in SiR-FP23 and native SiR-FP is compared to that of cytochro me P450 reductase and bacterial cytochrome P450, which also contain on e FAD and one FMN per polypeptide chain.