Insect Biochemistry and Molecular Biology 29 (1999) 233–242 Kinetic mechanism of cytochrome P450 reductase from the house fly (Musca domestica) Marat B. Murataliev, Adrienne Arin ˜o, Victor M. Guzov 1 , Rene ´ Feyereisen * Department of Entomology and Center for Toxicology, University of Arizona, Tucson, AZ 85721-0036, USA Received 18 August 1998; received in revised form 14 December 1998; accepted 15 December 1998 Abstract Recombinant house fly (Musca domestica) cytochrome P450 reductase has been purified by anion exchange and affinity chromato- graphy. 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 + (K d of 0.31 and 3.3 μM, 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.  1999 Elsevier Science Ltd. All rights reserved. Keywords: Cytochrome P450 reductase; Flavoprotein; Kinetic mechanism 1. Introduction Insect cytochrome P450 systems are known to be involved in numerous reactions of xenobiotic metab- olism including detoxification of insecticides, plant and microbial toxins, as well as in the biosynthesis of insect hormones (Feyereisen, 1999). NADPH cytochrome P450 reductase (EC 1.6.2.4) plays a key role in microsomal cytochrome P450-dependent monooxygenase reactions. Alone or in conjunction with cytochrome b 5 , it provides two electrons to cytochrome P450 enzymes. The gene of the microsomal P450 reductase from the house fly, Musca domestica, has been cloned (Koener et al., 1993) Abbreviations: P450 reductase; NADPH-cytochrome P450 reductase; P450BM3; cytochrome P450BM3; isolated from Bacillus megaterium. * Corresponding author. Tel.: + 520 621 9598; fax: + 520 626 8058. E-mail address: rfeyer@ag.arizona.edu (R. Feyereisen) 1 Present address: Monsanto Company, 700 Chesterfield Parkway North, St. Louis, MO 63198. 0965-1748/99/$ - see front matter.  1999 Elsevier Science Ltd. All rights reserved. PII:S0965-1748(98)00131-3 and an expression system in E. coli developed in our laboratory (Andersen et al., 1994). P450 reductase is a flavoprotein with two flavin cofac- tors, FAD and FMN, as prosthetic groups. Reducing equivalents are transferred from NADPH to FAD, and delivered to a cytochrome P450 enzyme from FMN in two one-electron transfer steps. Mammalian P450 reductases have been extensively studied (for reviews see Gray, 1992; Backes, 1993; Masters and Okita, 1980; Peterson and Prough, 1986). Redox potentials of all half- reactions of the two flavins of the rat enzyme were determined (Iyanagi et al., 1974), and, based on these values, two models were proposed for the catalytic mechanism of P450 reductase. According to these mech- anisms, the mammalian enzyme cycles between either 1e - -3e - or 2e - -4e - reduction states during catalytic turnover, with fully reduced FMN (hydroquinone) believed to serve as the electron donor for cytochrome c or cytochrome P450 (see review by Backes, 1993). Identification of the kinetic mechanism of the enzyme is important for our understanding of the interaction with