Cm. Jenkins et Mr. Waterman, FLAVODOXIN AND NADPH-FLAVODOXIN REDUCTASE FROM ESCHERICHIA-COLI SUPPORT BOVINE CYTOCHROME P450C17 HYDROXYLASE-ACTIVITIES, The Journal of biological chemistry, 269(44), 1994, pp. 27401-27408
Two soluble flavoproteins, purified from Escherichia coli cytosol and
identified as flavodoxin and NADPH flavodoxin (ferredoxin) reductase (
flavodoxin reductase), have been found in combination to support the 1
7 alpha-hydroxylase activities of heterologously expressed bovine 17 a
lpha-hydroxylase cytochrome P450 (P450c17). Physical characteristics o
f the two flavoproteins including absorbance spectra, molecular weight
s, and amino terminal sequences are identical with those reported prev
iously for E. coli flavodoxin and flavodoxin reductase. Flavodoxin red
uctase, possessing FAD as a cofactor, is able to reconstitute P450c17
activities only in the presence of flavodoxin, an FMN containing prote
in, and NAD(P)H. Reducing equivalents are utilized more effectively fr
om NADPH than NADH by flavodoxin reductase. E. coli flavodoxin binds P
450c17 directly and with relatively high affinity (apparent K-s simila
r to 0.2 mu M) at low ionic strength, as evidenced by a change in spin
state of the P450c17 heme iron upon titration with flavodoxin. This a
pparent spin shift is attenuated at moderate ionic strengths (100-200
mM KCl). In addition, bovine P450c17 binds reversibly to flavodoxin Se
pharose in an ionic strength dependent manner. These data implicate ch
arge pairing as being important for the interaction between flavodoxin
and P450c17. We propose that the amino acid sequence similarity betwe
en E. coli flavodoxin-flavodoxin reductase and the putative FMN, FAD,
and NAD(P)H binding regions of cytochrome P450 reductase provides the
basis for the reconstitution of P450c17 activities by this bacterial s
ystem.