CHARACTERIZATION OF FLAVODOXIN NADP(- KEY COMPONENTS OF ELECTRON-TRANSFER IN ESCHERICHIA-COLI() OXIDOREDUCTASE AND FLAVODOXIN )

The genes encoding the Escherichia coli flavodoxin NADP(+) oxidoreduct ase (FLDR) and flavodoxin (FLD) have been overexpressed in E. coli as the major cell proteins (at least 13.5% and 11.4% of total soluble pro tein, respectively) and the gene products purified to homogeneity. The FLDR reduces potassium ferricyanide with a k(cat) of 1610.3 min(-1) a nd a K-m of 23.6 mu M, and cytochrome c with a k(cat) of 141.3 min(-1) and a K-m of 17.6 mu M. The cytochrome c reductase rate is increased sixfold by addition of FLD and an apparent K-m of 6.84 mu M was measur ed or the affinity of the two flavoproteins. The molecular masses of F LDR and FLD apoproteins were determined as 27648 Da and 19606 Da and t he isolectric points as 4.8 and 3.5, respectively. The mass of the FLD R is precisely that predicted from the atomic structure and indicates that residue 126 is arginine, not glutamine as predicted from the gene sequence. FLDR and FLD were covalently crosslinked using 1-ethyl-3(di methylamino-propyl) carbodiimide to generate a catalytically active he terodimer. The midpoint reduction potentials of the oxidised/semiquino ne and semiquinone/hydroquinone couples of both FLDR (-308 mV and -268 mV, respectively) and FLD (-254 mV and -433 mV, respectively) were me asured using redox potentiometry. This confirms the electron-transfer route as NADPH-->FLDR-->FLD. Binding of 2' adenosine monophosphate inc reases the midpoint reduction potentials for both FLDR couples. These data highlight the strong stabilisation of the flavodoxin semiquinone (absorption coefficient calculated as 4933 M-1 cm(-1) at 583 nm) with respect to the hydroquinone state and indicate that FLD must act as a Single electron shuttle from the semiquinone form in its support of ce llular functions, and to facilitate catalytic activity of microsomal c ytochromes P-450 heterologously expressed in E. coli. Kinetic studies of electron transfer from FLDR/FLD to the fatty acid oxidase P-450 BM3 support this conclusion, indicating a ping-pong mechanism. This is th e first report of the potentiometric analysis of the full E. coli NAD( P)H/FLDR/FLD electron-transfer chain; a complex critical to the functi on of a large number of E. coli redox systems.