THE FLAVOPROTEIN DOMAIN OF P450BM-3 - EXPRESSION, PURIFICATION, AND PROPERTIES OF THE FLAVIN ADENINE DINUCLEOTIDE-BINDING AND FLAVIN MONONUCLEOTIDE-BINDING SUBDOMAINS
I. Sevrioukova et al., THE FLAVOPROTEIN DOMAIN OF P450BM-3 - EXPRESSION, PURIFICATION, AND PROPERTIES OF THE FLAVIN ADENINE DINUCLEOTIDE-BINDING AND FLAVIN MONONUCLEOTIDE-BINDING SUBDOMAINS, Biochemistry, 35(23), 1996, pp. 7528-7535
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.