THE FLAVOPROTEIN DOMAIN OF P450BM-3 - EXPRESSION, PURIFICATION, AND PROPERTIES OF THE FLAVIN ADENINE DINUCLEOTIDE-BINDING AND FLAVIN MONONUCLEOTIDE-BINDING SUBDOMAINS

P450BM-3 is a self-sufficient fatty acid monooxygenase that can be exp ressed in Escherichia coli as either the holoenzyme or as the individu al hemo- and flavoprotein domains. The flavoprotein domain (BMR) of P4 50BM-3 is soluble and contains an equimolar ratio of flavin adenine di nucleotide (FAD) and flavin mononucleotide (FMN) and is functionally a nalogous to microsomal nicotinamide adenine dinucleotide phosphate (NA DPH)-P450 reductases. These reductases have been proposed to have evol ved through a fusion of genes encoding simple flavin-containing electr on-transport proteins [Porter, T. D. (1991) Trends Biochem. Sci. 16, 1 54-158]. The gene encoding BMR has been divided into the coding region s for the FAD/NADPH- and FMN-binding domains. These proteins were over expressed in E. coli and both domains were found to contain not less t han 0.9 +/- 0.05 mol of FAD or FMN/mol of protein. Compared to BMR, th e electron-accepting properties of the recombinant flavin domains were mainly conserved. Titration of the FMN domain with sodium dithionite resulted in the conversion of the protein to the fully reduced FMNH(2) form without accumulation of intermediate semiquinone forms; however, a similar titration of the FAD domain gave clear evidence for the pre sence of a neutral, blue flavin semiquinone during the reduction. Titr ations of the reduced forms of the domains with artificial electron ac cepters indicated that the electron-transferring properties of both th e FAD- and FMN domains were also conserved. The rate constants of reox idation of the fully reduced FAD and FMN domains by molecular oxygen a t 20 degrees C were found to be 2.5 and 0.1 min(-1), respectively. The cytochrome c reductase activity of BMR could be fully reconstituted w ith the individual domains. The data presented support the hypothesis that BMR has a discrete multidomain structure.