Separate roles for FMN and FAD in catalysis by liver microsomal NADPH-cytochrome P-450 reductase

• Published in: The Journal of biological chemistry Vol. 256; no. 1; pp. 266 - 277
• Main Authors: Vermilion, J L, Ballou, D P, Massey, V, Coon, M J
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 10-01-1981
• Subjects: Animals; Flavin Mononucleotide - metabolism; Flavin-Adenine Dinucleotide - metabolism; Flavins - pharmacology; Kinetics; Microsomes, Liver - enzymology; NADPH-Ferrihemoprotein Reductase - metabolism; Oxidation-Reduction; Rats; Spectrophotometry
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(19)70129-x

Rat liver microsomal NADPH-cytochrome P-450 reductase was prepared free of detectable amounts of FMN by a new procedure based
on the exchange of this flavin into apoflavodoxin. The resulting FMN-free reductase binds NADP in the oxidized state with
the same affinity (Kd = 5 microM) and stoichiometry (1:1 molar ratio) as does the native enzyme. Both the native and FMN-free
reductase catalyze rapid reduction of ferricyanide, but the ability to reduce th 5,6-benzoflavone-inducible form of the liver
microsomal cytochrome P-450 (P-450LM4) is lost upon removal of FMN. The FMN-free enzyme was reconstituted with artificial
flavins which, in the free state, have oxidation-reduction potentials ranging from -152 to -290 mV, including 5-carba-5-deaza-FMN
and several FMN analogs with a halogen or sulfur substituent on the dimethylbenzene portion of the ring system. Enzyme reconstituted
with 5-carba-5-deaza-FMN has catalytic properties which are not significantly different from those of the FMN-free reductase,
and is unable to reduce P-450LM4. On the other hand, the ability to reduce P-450LM4 and the other FMN-dependent activities
of the native reductase are restored by substitution of several other analogs for FMN, but the kinetics of P-450LM4 reduction,
studied under anaerobic conditions by stopped flow spectrophotometry, are significantly altered. The oxidation-reduction behavior
of enzyme reconstituted with 7-nor-7-Br-FMN is substantially different from that of the native enzyme, and less thermodynamic
stabilization of the semiquinone is observed with this flavin analog. In contrast, the oxidation-reduction properties of enzyme
containing 8-nor-8-mercapto-FMN are similar to those of the native enzyme, but the spectral properties are significantly different.
As shown in a stopped flow experiment, reduction of this FMN analog precedes reduction of P-450LM4 when a complex of the flavoprotein
and P-450LM4 is allowed to react with NADPH. Our experiments support a sequence of electron transfer in this enzyme system
as follows: NADPH leads to FAD leads to FMN leads to P-450. We propose that the enzyme cycles between a le- and a 3e-reduced
state during turnover and that electrons are donated to acceptors via the reaction, FMNH2 leads to FMNH ..