Insect Biochem Mol Biol. 1999 Mar;29(3):233-42.  

Kinetic mechanism of cytochrome P450 reductase from the house fly (Musca
domestica).

Murataliev MB, Arino A, Guzov VM, Feyereisen R.

Department of Entomology, University of Arizona, Tucson 85721-0036, USA.

Recombinant house fly (Musca domestica) cytochrome P450 reductase has been
purified by anion exchange and affinity chromatography. Steady-state kinetics of
cytochrome c reductase activity revealed a random Bi-Bi mechanism with formation
of a ternary P450 reductase-NADPH-electron acceptor complex as catalytic
intermediate. NADP(H) binding is essential for fast hydride ion transfer to FAD,
as well as for electron transfer from FMN to cytochrome c. Reduced cytochrome c
had no effect on the enzyme activity, while NADP+ and 2'-AMP inhibited P450
reductase competitively with respect to NADPH and noncompetitively with respect
to cytochrome c. The affinity of the P450 reductase to NADPH is 10 times higher
than to NADP+ (Kd of 0.31 and 3.3 microM, respectively). Such an affinity change
during catalysis could account for a +30 mV shift of the redox potential of FAD.
Cys560 was substituted for Tyr by site-directed mutagenesis. This mutation
decreased enzyme affinity to NADPH 35-fold by decreasing the bimolecular rate
constant of nucleotide binding with no detectable effect on the kinetic
mechanism. The affinity of the C560Y mutant enzyme to NADP+ decreased 9-fold
compared to the wild-type enzyme, while the affinity to 2'-AMP was not
significantly affected, suggesting that Cys560 is located in the nicotinamide
binding site of the active, full-size enzyme in solution.

PMID: 10319436 [PubMed - indexed for MEDLINE]