Site-Directed Mutagenesis of Cytochrome P450scc (CYP11A1). Effect of Lysine Residue Substitution on Its Structural and Functional Properties

Our previous chemical modification and cross-linking studies identified some positively charged amino acid residues of cytochrome P450scc that may be important for its interaction with adrenodoxin and for its functional activity. The present study was undertaken to further evaluate the role of these residues in the interaction of cytochrome P450scc with adrenodoxin using site-directed mutagenesis. Six cytochrome P450scc mutants containing replacements of the surface-exposed positively charged residues (Lys103Gln, Lys110Gln, Lys145Gln, Lys394Gln, Lys403Gln, and Lys405Gln) were expressed in E. coli cells, purified as a substrate-bound high-spin form, and characterized as compared to the wild-type protein. The replacement of the surface Lys residues does not dramatically change the protein folding or the heme pocket environment as judged from limited proteolysis and spectral studies of the cytochrome P450 mutants. The replacement of Lys in the N-terminal sequence of P450scc does not dramatically affect the activity of the heme protein. However, mutant Lys405Gln revealed rather dramatic loss of cholesterol side-chain cleavage activity, efficiency of enzymatic reduction in a reconstituted system, and apparent dissociation constant for adrenodoxin binding. The present results, together with previous findings, suggest that the changes in functional activity of mutant Lys405Gln may reflect the direct participation of this amino acid residue in the electrostatic interaction of cytochrome P450scc with its physiological partner, adrenodoxin.     

Role of Positively Charged Residues Lys267, Lys270, and Arg411 of Cytochrome P450scc (Cyp11A1) in Interaction with Adrenodoxin

Cytochrome P450scc and adrenodoxin are redox proteins of the electron transfer chain of the inner mitochondrial membrane steroid hydroxylases. In the present work site-directed mutagenesis of the charged residues of cytochrome P450scc and adrenodoxin, which might be involved in interaction, was used to study the nature of electrostatic contacts between the hemeprotein and the ferredoxin. The target residues for mutagenesis were selected based on the theoretical model of cytochrome P450scc-adrenodoxin complex and previously reported chemical modification studies of cytochrome P450scc. In the present work, to clarify the molecular mechanism of hemeprotein interaction with ferredoxin, we constructed cytochrome P450scc Lys267, Lys270, and Arg411 mutants and Glu47 mutant of adrenodoxin and analyzed their possible role in electrostatic interaction and the role of these residues in the functional activity of the proteins. Charge neutralization at positions Lys267 or Lys270 of cytochrome P450scc causes no significant effect on the physicochemical and functional properties of cytochrome P450scc. However, cytochrome P450scc mutant Arg411Gln was found to exhibit decreased binding affinity to adrenodoxin and lower activity in the cholesterol side chain cleavage reaction. Studies of the functional properties of Glu47Gln and Glu47Arg adrenodoxin mutants indicate that a negatively charged residue in the loop covering the Fe2S2 cluster, being important for maintenance of the correct architecture of these structural elements of ferredoxin, is not directly involved in electrostatic interaction with cytochrome P450scc. Moreover, our results indicate the presence of at least two different binding (contact) sites on the proximal surface of cytochrome P450scc with different electrostatic input to interaction with adrenodoxin. In the binary complex, the positively charged sites of the proximal surface of cytochrome P450scc well correspond to the two negatively charged sites of adrenodoxin: the "interaction" domain site and the "core" domain site.     

Site-Directed Mutagenesis of Cytochrome P450scc. II. Effect of Replacement of the Arg425 and Arg426 Residues on the Structural and Functional Properties of the Cytochrome P450scc

Cytochrome P450-dependent monooxygenases, in spite of their wide distribution, can be simply divided into a few groups differing in the location of the electron transfer chain and their composition. The two main groups of cytochrome P450-dependent monooxygenases are the mitochondrial and microsomal enzymes. While in two-component microsomal cytochrome P450-dependent monooxygenases electrons are supplied to cytochrome P450 by a flavoprotein (NADPH-cytochrome P450 reductase), in three-component mitochondrial monooxygenases the electrons are supplied to cytochrome P450 by a low molecular weight protein (ferredoxin). The interaction of cytochrome P450 with NADPH-cytochrome P450 reductase and ferredoxin is the subject of intensive studies. Using chemical modification, chemical cross-linking, and sitedirected mutagenesis, we identified surface exposed positively charged residues of cytochrome P450scc which might be important for interaction with adrenodoxin. Theoretical analysis of the distribution of surface electrostatic potential in cytochrome P450 indicates that in contrast to microsomal monooxygenases, cytochromes P450 of mitochondrial type, and cholesterol side-chain cleavage cytochrome P450 (P450scc) in part, carry on the proximal surface an evidently positively charged site that is formed by residues Arg425 and Arg426. In the present work, to estimate the functional role of Arg425 and Arg426 of cytochrome P450scc, we used site-directed mutagenesis to replace these residues with glutamine. The results indicate that residues Arg425 and Arg426 are involved in the formation of a heme-binding center and electrostatic interaction of cytochrome P450scc with its physiological electron-transfer partner, adrenodoxin.     

The interaction of microsomal cytochrome P450 2B4 with its redox partners, cytochrome P450 reductase and cytochrome b(5)

Cytochrome P450 2B4 is a microsomal protein with a multi-step reaction cycle similar to that observed in the majority of other cytochromes P450. The cytochrome P450 2B4-substrate complex is reduced from the ferric to the ferrous form by cytochrome P450 reductase. After binding oxygen, the oxyferrous protein accepts a second electron which is provided by either cytochrome P450 reductase or cytochrome b₅. In both instances, product formation occurs. When the second electron is donated by cytochrome b₅, catalysis (product formation) is ∼10- to 100-fold faster than in the presence of cytochrome P450 reductase. This allows less time for side product formation (hydrogen peroxide and superoxide) and improves by ∼15% the coupling of NADPH consumption to product formation. Cytochrome b₅ has also been shown to compete with cytochrome P450 reductase for a binding site on the proximal surface of cytochrome P450 2B4. These two different effects of cytochrome b₅ on cytochrome P450 2B4 reactivity can explain how cytochrome b₅ is able to stimulate, inhibit, or have no effect on cytochrome P450 2B4 activity. At low molar ratios (<1) of cytochrome b₅ to cytochrome P450 reductase, the more rapid catalysis results in enhanced substrate metabolism. In contrast, at high molar ratios (>1) of cytochrome b₅ to cytochrome P450 reductase, cytochrome b₅ inhibits activity by binding to the proximal surface of cytochrome P450 and preventing the reductase from reducing ferric cytochrome P450 to the ferrous protein, thereby aborting the catalytic reaction cycle. When the stimulatory and inhibitory effects of cytochrome b₅ are equal, it will appear to have no effect on the enzymatic activity. It is hypothesized that cytochrome b₅ stimulates catalysis by causing a conformational change in the active site, which allows the active oxidizing oxyferryl species of cytochrome P450 to be formed more rapidly than in the presence of reductase.     

Cytochrome b5 involvement in cytochrome P450 monooxygenase activities in house fly microsomes

The involvement of cytochrome b₅ in different cytochrome P450 monooxygenase and palmitoyl CoA desaturase activities in microsomes from insecticide-resistant (LPR) house flies was determined using a specific polyclonal antiserum developed against house fly cytochrome b₅. Anti-b₅ antiserum inhibited the reduction of cytochrome b₅ by NADH-cytochrome b₅ reductase. The antiserum also inhibited palmitoyl CoA desaturase, methoxycoumarin-O-demethylase (MCOD), ethoxycoumarin-O-deethylase (ECOD), and benzo[a]pyrene hydroxylase (aromatic hydrocarbon hydroxylase, AHH) activities. However, methoxyresorufin-O-demethylase (MROD) and ethoxyresorufin-O-deethy-lase (EROD) activities were not affected by this antiserum. These results demonstrate that cytochrome b₅ is involved in fatty acyl CoA desaturase activities and in certain cytochrome P450 monooxygenase activities (i.e., MCOD, ECOD, and AHH) in LPR house fly microsomes. Other cytochrome P450 monooxygenase activities (i.e., MROD and EROD) may not require cytochrome b₅. The results suggest that cytochrome b₅ involvement with cytochrome P450 monooxygenase activities is dependent upon the cytochrome P450 isoform involved. © 1994 Wiley-Liss, Inc.     

Involvement of cytochrome b5 in the cytotoxic response to Escherichia coli Lipopolysaccharide

Cytotoxic lesions, induced by Gram-negative lipopolysaccharides (LPS), occur mainly in liver where the microsomal compartment of hepatocytes is involved in the detoxification mechanisms as well as in the biosynthesis of different active metabolites.The alterations induced by LPS from E. coli 0111: 134 on cytochrome b₅ and its correlation with cytochrome P450, have been studied using an in vivo reversible endotoxic shock model and 24 h non-replicative hepatocyte monolayers.Results show that cytochrome b₅ is directly affected by LPS that induces also a membrane damage with an active release of lactate dehydrogenase (LDH). The increase of cytochrome b₅ levels may enhance the efficiency of the electron transport, thus facilitating the cytochrome P450-associate oxidations and reactions involved in the repair mechanisms of membranes.     

Cytochrome b5 reductase–cytochrome b5 as an active P450 redox enzyme system in Phanerochaete chrysosporium: Atypical properties and in vivo evidence of electron transfer capability to CYP63A2

Two central redox enzyme systems exist to reduce eukaryotic P450 enzymes, the P450 oxidoreductase (POR) and the cyt b₅ reductase–cyt b₅. In fungi, limited information is available for the cyt b₅ reductase–cyt b₅ system. Here we characterized the kinetic mechanism of (cyt b₅r)–cyt b₅ redox system from the model white-rot fungus Phanerochaete chrysosporium (Pc) and made a quantitative comparison to the POR system. We determined that Pc-cyt b₅r followed a “ping-pong” mechanism and could directly reduce cytochrome c. However, unlike other cyt b₅ reductases, Pc-cyt b₅r lacked the typical ferricyanide reduction activity, a standard for cyt b₅ reductases. Through co-expression in yeast, we demonstrated that the Pc-cyt b₅r–cyt b₅ complex is capable of transferring electrons to Pc-P450 CYP63A2 for its benzo(a)pyrene monooxygenation activity and that the efficiency was comparable to POR. In fact, both redox systems supported oxidation of an estimated one-third of the added benzo(a)pyrene amount. To our knowledge, this is the first report to indicate that the cyt b₅r–cyt b₅ complex of fungi is capable of transferring electrons to a P450 monooxygenase. Furthermore, this is the first eukaryotic quantitative comparison of the two P450 redox enzyme systems (POR and cyt b₅r–cyt b₅) in terms of supporting a P450 monooxygenase activity.     

Sunflower cDNA Encoding New Cytochrome P450

The primary structure of the cDNA clone SF28 was determined in sunflower (Helianthus annuusL.) flowers. The clone comprises a 874-bp insert corresponding to 227 amino acid residues of the C-terminal part of the cytochrome P450 gene. The sunflower cytochrome P450 was considerably different from the already known plant and animal cytochromes P450.     

Substrate-modulated Cytochrome P450 17A1 and Cytochrome b5 Interactions Revealed by NMR

The membrane heme protein cytochrome b₅ (b₅) can enhance, inhibit, or have no effect on cytochrome P450 (P450) catalysis, depending on the specific P450, substrate, and reaction conditions, but the structural basis remains unclear. Here the interactions between the soluble domain of microsomal b₅ and the catalytic domain of the bifunctional steroidogenic cytochrome P450 17A1 (CYP17A1) were investigated. CYP17A1 performs both steroid hydroxylation, which is unaffected by b₅, and an androgen-forming lyase reaction that is facilitated 10-fold by b₅. NMR chemical shift mapping of b₅ titrations with CYP17A1 indicates that the interaction occurs in an intermediate exchange regime and identifies charged surface residues involved in the protein/protein interface. The role of these residues is confirmed by disruption of the complex upon mutagenesis of either the anionic b₅ residues (Glu-48 or Glu-49) or the corresponding cationic CYP17A1 residues (Arg-347, Arg-358, or Arg-449). Cytochrome b₅ binding to CYP17A1 is also mutually exclusive with binding of NADPH-cytochrome P450 reductase. To probe the differential effects of b₅ on the two CYP17A1-mediated reactions and, thus, communication between the superficial b₅ binding site and the buried CYP17A1 active site, CYP17A1/b₅ complex formation was characterized with either hydroxylase or lyase substrates bound to CYP17A1. Significantly, the CYP17A1/b₅ interaction is stronger when the hydroxylase substrate pregnenolone is present in the CYP17A1 active site than when the lyase substrate 17α-hydroxypregnenolone is in the active site. These findings form the basis for a clearer understanding of this important interaction by directly measuring the reversible binding of the two proteins, providing evidence of communication between the CYP17A1 active site and the superficial proximal b₅ binding site.     

The cytochromes P450 and b5 and their reductases—Promising targets for structural studies by advanced solid-state NMR spectroscopy

Members of the cytochrome P450 (cyt P450) superfamily of enzymes oxidize a wide array of endogenous and xenobiotic substances to prepare them for excretion. Most of the drugs in use today are metabolized in part by a small set of human cyt P450 isozymes. Consequently, cyt P450s have for a long time received a lot of attention in biochemical and pharmacological research. Cytochrome P450 receives electrons from cytochrome P450 reductase and in selected cases from cytochrome b₅ (cyt b₅). Numerous structural studies of cyt P450s, cyt b₅, and their reductases have given considerable insight into fundamental structure-function relationships. However, structural studies so far have had to rely on truncated variants of the enzymes to make conventional X-ray crystallographic and solution-state NMR techniques applicable. In spite of significant efforts it has not yet been possible to crystallize any of these proteins in their full-length membrane bound forms. The truncated parts of the enzymes are assumed to be α-helical membrane anchors that are essential for some key properties of cyt P450s. In the present contribution we set out with a basic overview on the current status of functional and structural studies. Our main aim is to demonstrate how advanced modern solid-state NMR spectroscopic techniques will be able to make substantial progress in cyt P450 research. Solid-state NMR spectroscopy has sufficiently matured over the last decade to be fully applicable to any membrane protein system. Recent years have seen a remarkable increase in studies on membrane protein structure using a host of solid-state NMR techniques. Solid-state NMR is the only technique available today for structural studies on full-length cyt P450 and full-length cyt b₅. We aim to give a detailed account of modern techniques as applicable to cyt P450 and cyt b₅, to show what has already been possible and what seems to be viable in the very near future.     

Role of C-terminal sequence of cytochrome P450scc in folding and functional activity

To elucidate the role of Arg472 and C-terminal sequence of the mature form of cytochrome P450scc, a mitochondrial cytochrome P450, in the present work we have performed sequential removal of the C-terminal amino acid residues of the hemeprotein and evaluated their functional role in folding and catalysis. The removal of 2, 4, 7, or 9 amino acid residues (cytochrome P450scc mutants Delta2, Delta4, Delta7, and Delta9) does not significantly affect the physicochemical properties of the truncated forms of cytochrome P450scc, but results in significant increase in the expression level of the hemeprotein in Escherichia coli (Delta4 cytochrome P450scc mutant). However, removal of 10 C-terminal amino acid residues (Delta10 cytochrome P450scc) of mature form of cytochrome P450scc (replacement of codon for Arg472 for stop-codon) is followed by loss of the ability for correct folding in E. coli. Based on these data, it is concluded that the C-terminal amino acid residues of cytochrome P450scc (DeltaArg472-Ala481) play an important role in correct recombinant protein folding and heme binding by cytochrome P450scc during its expression in E. coli, while folding of mitochondrial cytochrome P450scc during its heterologous expression in bacterial cells is more similar to the folding of prokaryotic soluble cytochrome P450's than to microsomal cytochrome P450's.     

Productive and non-productive complexes in cytochrome P450-containing systems

The equilibrium dissociation constants K_D, the complex association / dissociation rate constants (k _on /k _off) and lifetimes of the complexes of redox partners were measured for three cytochrome P450-containing monooxygenase systems (P450cam, P450scc, and P450 2B4) under hydroxylation conditions. The Q parameter representing the ratio of protein-protein complex lifetime (τ _lT ) to time required for a single hydroxylation cycle (τ_turnover) was introduced for estimation of productivity of complexes formed within the systems studied. The Q parameter was insignificantly changed upon transition from the oxidation to hydroxylation conditions. Lifetimes (τ _lT ) for the binary complexes formed within the P450cam and the P450scc systems obligatory requiring an intermediate electron transfer protein between the reductase and cytochrome P450 could not realize hydroxylation reactions for substrates with known τ_turnover and so they were non-productive while the binary complexes formed within the P450 2B4 system, not requiring such intermediate electron-transfer protein, appeared to be productive. Formation of ternary complexes was demonstrated under hydroxylation conditions in all three systems. Analysis of Q values led to the conclusion that the ternary complexes formed within the P450cam and the P450scc systems were productive. In the case of the P450 2B4 system, more than half (about 60%) ternary complexes were also found to be productive.     

Expression of human cytochrome p450 3A4 gene in Schizosaccharomyces pombe

Heterologous expression systems can be utilized to great advantage in the study of cytochrome P450 enzymes. P450 3A4 is one of the major forms of cytochrome P450 found in liver. It is also involved in the metabolism of numerous widely used drugs and xenobiotics. In the present study human liver cytochrome P450 3A4 gene was transferred into the fission yeast Schizosaccharomyces pombe via two different S. pombe expression vectors carrying thiamine repressible promoter — nmt1 (pREP42) and constitutive promoter — adh1 (pART1). Heterologously expressed cytochrome P450 3A4 was detected in the cells grown in minimal (EMM) or rich medium (YEL) containing 0.5% (w/v) glucose. A typical cytochrome P450 peak for 3A4 was observed at 448 nm in microsomal fraction. The presence of heterologous expression of 3A4 form was also determined by SDS-PAGE and it molecular mass was identified as 52 kDa. The enzyme activity was confirmed by HPLC analysis, using testosterone as substrate.     

Human colon tumor cell line LS174T drug metabolizing system

The effects of culture variables on the specific content and activity of various enzymes of the drug mmetabolizing system were assessed in colon tumor cell line LS174T. The NADH reduced cytochrome b₅ (cyt b₅)⁴ spectrum of these cells was similar to rat liver cyt b₅. When released from the membrane by trypsin and concentrated, the cyt b₅ was found to cross react with rabbit antibody to rat liver cyt b₅ and human liver cyt b₅. The enzyme activities were found stable over limited cell passages with control values of 0.03 and 0.13 µol/min/mg protein for NADPH and NADH cytochrome c (cyt c) reducing activity, 0.05 nmol cyt b₅ and 0.013 nmol cytochrome P450 per milligram of microsomal protein. Phenobarbital/hydrocortisone showed a consistent, but not always significant increase in the NADPH and NADH cyt c reduction and benzanthracene an increase in the NADH cyt c reducing activity and cyt b₅ content. Griseofulvin lowered the NADH cyt c reducing activity. Delta-aminolevulinic acid (0.5 mM) caused a significant decrease in the specific activity of all enzymes, as judged by a student's t test, with a p<0.001.Abbreviations cyt b₅ cytochrome b₅ - cyt c cytochrome c - cyt P450 cytochrome P450 - PB Phenobarbital - HC Hydrocortisone - ALA -Aminolevulinic acid - GRIS Griseofulvin - PENT Pentagastrin - PASS Cell Passage - DMH Dimethylhydrazine - BA Benzanth Acene     

Effects of Membrane Mimetics on Cytochrome P450-Cytochrome b5 Interactions Characterized by NMR Spectroscopy

Mammalian cytochrome P450 (P450) is a membrane-bound monooxygenase whose catalytic activities require two electrons to be sequentially delivered from its redox partners: cytochrome b₅ (cytb₅) and cytochrome P450 reductase, both of which are membrane proteins. Although P450 functional activities are known to be affected by lipids, experimental evidence to reveal the effect of membrane on P450-cytb₅ interactions is still lacking. Here, we present evidence for the influence of phospholipid bilayers on complex formation between rabbit P450 2B4 (CYP2B4) and rabbit cytb₅ at the atomic level, utilizing NMR techniques. General line broadening and modest chemical shift perturbations of cytb₅ resonances characterize CYP2B4-cytb₅ interactions on the intermediate time scale. More significant intensity attenuation and a more specific protein-protein binding interface are observed in bicelles as compared with lipid-free solution, highlighting the importance of the lipid bilayer in stabilizing stronger and more specific interactions between CYP2B4 and cytb₅, which may lead to a more efficient electron transfer. Similar results observed for the interactions between CYP2B4 lacking the transmembrane domain (tr-CYP2B4) and cytb₅ imply interactions between tr-CYP2B4 and the membrane surface, which might assist in CYP2B4-cytb₅ complex formation by orienting tr-CYP2B4 for efficient contact with cytb₅. Furthermore, the observation of weak and nonspecific interactions between CYP2B4 and cytb₅ in micelles suggests that lipid bilayer structures and low curvature membrane surface are preferable for CYP2B4-cytb₅ complex formation. Results presented in this study provide structural insights into the mechanism behind the important role that the lipid bilayer plays in the interactions between P450s and their redox partners.     

A Model of the Membrane-bound Cytochrome b5-Cytochrome P450 Complex from NMR and Mutagenesis Data

Microsomal cytochrome b₅ (cytb₅) is a membrane-bound protein that modulates the catalytic activity of its redox partner, cytochrome P4502B4 (cytP450). Here, we report the first structure of full-length rabbit ferric microsomal cytb₅ (16 kDa), incorporated in two different membrane mimetics (detergent micelles and lipid bicelles). Differential line broadening of the cytb₅ NMR resonances and site-directed mutagenesis data were used to characterize the cytb₅ interaction epitope recognized by ferric microsomal cytP450 (56 kDa). Subsequently, a data-driven docking algorithm, HADDOCK (high ambiguity driven biomolecular docking), was used to generate the structure of the complex between cytP4502B4 and cytb₅ using experimentally derived restraints from NMR, mutagenesis, and the double mutant cycle data obtained on the full-length proteins. Our docking and experimental results point to the formation of a dynamic electron transfer complex between the acidic convex surface of cytb₅ and the concave basic proximal surface of cytP4502B4. The majority of the binding energy for the complex is provided by interactions between residues on the C-helix and β-bulge of cytP450 and residues at the end of helix α4 of cytb₅. The structure of the complex allows us to propose an interprotein electron transfer pathway involving the highly conserved Arg-125 on cytP450 serving as a salt bridge between the heme propionates of cytP450 and cytb₅. We have also shown that the addition of a substrate to cytP450 likely strengthens the cytb₅-cytP450 interaction. This study paves the way to obtaining valuable structural, functional, and dynamic information on membrane-bound complexes.     

The cytochrome P450-containing monooxygenase of Trichosporon cutaneum: Occurrence and properties

Trichosporon cutaneum metabolizes glucose purely oxidatively and cytochrome P450 was not detected in the reduced CO-difference spectrum of whole cells. However, in the isolated microsomal fraction the corresponding monooxygenase was present as shown by the appearence of cytochrome P450, NADPH-cytochrome c (P450) reductase and cytochrome b₅. The absorption maximum of the terminal oxidase in the reduced CO-difference spectrum shifted between 447 and 448 nm. Derepression of biosynthesis of all components was achieved by transition of the cells from carbon- to oxygen-limited growth in continuous culture. The monooxygenase exhibited aminopyrine demethylation activity but not -hydroxylation activity of lauric acid. With respect to the growth limiting nutrient (carbon and oxygen respectively), mitochondrial cytochrome content showed an analogous behavior as cytochrome P450 and cytochrome b₅.     

Expression of cytochrome P450scc in Escherichia coli cells: Intracellular location and interaction with bacterial redox proteins

Escherichia coli cells producing the mature form of adrenal cytochrome P450scc were used as a model for study of cytochrome P450scc topogenesis. By disruption of transformed E. coli cells and centrifugation of the homogenate under conventional conditions, we obtained membrane and soluble (high-speed supernatant) fractions both containing the recombinant protein. Gel-permeation high performance liquid chromatography showed that in the high-speed supernatant the native cytochrome P450scc exists exclusively as a component of membrane fragments exceeding 400 kD. These data supported by kinetic assays suggest that the >400-kD particles containing P450scc are lipoprotein associates. In total, we failed to detect a genuine soluble cytochrome P450scc in the E. coli cells, which suggests that membrane insertion is an obligatory stage of holoenzyme formation. In the high-speed supernatant supplemented with NADPH, cytochrome P450scc underwent one-electron reduction and could convert 22R-hydroxycholesterol into pregnenolone. Thus, we have for the first time observed functional coupling of cytochrome P450scc with the bacterial electron transfer system.     

Enhancement of Isoflavone Synthase Activity by Co-expression of P450 Reductase from Rice

Plant cytochrome P450s interact with a flavoprotein, NADPH-cytochrome P450 reductase (CPR), to transfer electrons from NADPH. The gene for rice P450 reductase (RCPR) was cloned and expressed in Saccaromyces cerevisiae, where the specific activity of the expressed RPCR was 0.91 U/mg protein. When isoflavone synthase gene (IFS) from red clover, used as a model system of plant cytochrome P450, was co-expressed with RCPR in yeast, the production of genistein from naringein increased about 4.3-fold, indicating that the RCPR efficiently interacts with cytochrome P450 to transfer electrons from NADPH.