Justicidin B 7-hydroxylase, a cytochrome P450 monooxygenase from cell cultures of Linum perenne Himmelszelt involved in the biosynthesis of diphyllin

Cell suspension cultures of Linum perenne L. Himmelszelt accumulate justicidin B as the main component together with glycosides of 7-hydroxyjusticidin B (diphyllin). A hypothetical biosynthetic pathway for these compounds is suggested. Justicidin B 7-hydroxylase (JusB7H) catalyzes the last step in the biosynthesis of diphyllin by introducing a hydroxyl group in position 7 of justicidin B. This enzyme was characterized from a microsomal fraction prepared from a Linum perenne Himmelszelt suspension culture for the first time. The hydroxylase activity was strongly inhibited by cytochrome c as well as other cytochrome P450 inhibitors like clotrimazole indicating the involvement of a cytochrome P450-dependent monooxygenase. JusB7H has a pH optimum of 7.4 and a temperature optimum of 26 degrees C. Justicidin B was the only substrate accepted by JusB7H with an apparent K(m) of 3.9+/-1.3 microM. NADPH is predominantly accepted as the electron donor, but NADH was a weak co-substrate. A synergistic effect of NADPH and NADH was not observed. The apparent K(m) for NADPH is 102+/-10 microM.     

Deoxypodophyllotoxin 6-hydroxylase, a cytochrome P450 monooxygenase from cell cultures of Linum flavum involved in the biosynthesis of cytotoxic lignans

Cell-suspension cultures of Linum flavum L. (Linaceae) synthesize and accumulate aryltetrahydronaphthalene lignans with 6-methoxypodophyllotoxin as the main component. The experimental data indicate that the biosynthesis of 6-methoxypodophyllotoxin occurs via deoxypodophyllotoxin, beta-peltatin, and beta-peltatin-A methyl ether. The enzyme catalyzing the introduction of the hydroxyl group in position 6 is deoxypodophyllotoxin 6-hydroxylase (DOP6H). The enzyme was shown to be a cytochrome P450-dependent monooxygenase by blue-light reversion of carbon monoxide inhibition and inhibition by cytochrome c. DOP6H is a membrane-bound microsomal enzyme with a pH optimum of 7.6 and a temperature optimum of 26 degrees C. Deoxypodophyllotoxin is specifically accepted with an apparent Km of 20 microM and a saturation concentration of 200 microM; 4'-demethyldeoxypodophyllotoxin is the only other tested substrate accepted for hydroxylation. DOP6H predominantly accepts NADPH as electron donor; NADH can only sustain low hydroxylation activities. A synergistic effect of NADPH and NADH is not observed. The enzyme is saturated around 250 microM NADPH; the apparent Km for this substrate is 36 microM.     

8-dimethylallylnaringenin 2'-hydroxylase, the crucial cytochrome P450 mono-oxygenase for lavandulylated flavanone formation in Sophora flavescens cultured cells.

8-dimethylallylnaringenin (8-DMAN) 2'-hydroxylase, which is indispensable for the formation of a lavandulylated flavanone, sophoraflavanone G, was detected in cell suspension cultures of Sophora flavescens. The enzyme catalyzes the 2'-hydroxylation of 8-DMAN to leachianone G, and is tightly bound to the membrane. It required NADPH and molecular oxygen as cofactors, and was inhibited by several cytochrome P450 inhibitors such as carbon monoxide and cytochrome c, indicating that the reaction is mediated by a cytochrome P450 monooxygenase. The optimum pH of 8-DMAN 2'-hydroxylase was 8.5, and the enzyme hydroxylated only 8-DMAN. Apparent Km values for 8-DMAN and NADPH of the enzyme were 55 and 34 microM, respectively.     

Cinnamic acid 4-hydroxylase from cell cultures of the hornwort<Emphasis Type="Italic"> Anthoceros agrestis</Emphasis>

Cinnamic acid 4-hydroxylase (EC 1.14.13.11), a cytochrome P450-dependent hydroxylase was for the first time characterized from a hornwort, Anthoceros agrestis Paton (Anthocerotaceae). In suspension cultures of A. agrestis up to 5% of the dry weight was accumulated as rosmarinic acid, a natural product commonly known from higher plants (e.g. species of the Lamiaceae and Boraginaceae). Cinnamic acid 4-hydroxylase is involved in the biosynthesis of rosmarinic acid. The participation of cytochrome P450 was demonstrated by the inhibition of hydroxylase activity by cytochrome c and the inhibition of cinnamic acid hydroxylation in a CO-containing atmosphere, which is partially released by illumination with blue light. The apparent K(m) values were determined to be at 60 microM and 5 microM for NADPH and cinnamic acid, respectively. A comparatively high hydroxylation activity was seen with NADH as electron donor. While the hydroxylase activity with NADPH was strongly inhibited by the competitive electron acceptor cytochrome c, the activity with NADH was less susceptible, indicating the possibility of different electron-transfer pathways.     

Purification and partial characterization of NADPH-cytochrome P450 reductase from Gentiana triflora flowers

Reduced form of nicotineamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase was solubilized from a microsomal fraction of Gentiana triflora flowers by 3-[(3 Cholamidopropyl)-dimethylammonio]-1-propane sulfonate detergent and purified to electrophoretic homogeneity. The purification was achieved by adenosine 2', 5'-bisphosphate-Sepharose chromatography, followed by high-performance anion-exchange chromatography. A Mr value of 82,000 was obtained by SDS/polyacrylamide-gel electrophoresis. Western blot analysis showed that the purified protein cross-reacted with polyclonal antibody raised against rabbit anti-Gentiana triflora NADPH-cytochrome P450 reductase antibodies. The temperature and pH optimum for reduction of cytochrome c was 25 degrees C and 7.4 respectively. The Km values for the binding of NADPH and cytochrome c were 9.4 and 3.2 microM, respectively. In this paper, we present some results of the purification and partial characterization of microsomal NADPH-cytochrome P450 reductase from Gentiana triflora flowers.     

Lung microsomal p-nitrophenol hydroxylase -- characterization and reconstitution of its activity

1. Formation of catechols from benzene and nitrobenzene have been implicated in the carcinogenic activity of these chemicals. In liver, p-nitrophenol, an intermediate of p-nitrobenzene is enzymatically converted to 4-nitrocatechol. 2. For the first time in this study, the presence of a highly active enzyme catalyzing the formation of 4-nitrocatechol from p-nitrophenol was detected in lung microsomes. The average specific activity of lung p-nitrophenol hydroxylase was found to be 0.494 nmol 4-nitrocatechol formed mg prot-1 min-1. 3. The optimum conditions for sheep lung microsomal p-nitrophenol hydroxylase were established. The maximal activity was noted at pH 6.8. The rate of p-nitrophenol hydroxylation was linear up to 2 mg prot/ml of incubation mixture. The maximal rate of 4-nitrocatechol formation was observed with 0.25 mM p-nitrophenol. 4. The Lineweaver-Burk and Eadie-Hofstee plots were found to be curve-linear. Two different Km values were calculated as 11.6 and 71.4 microM from the Lineweaver-Burk plot and as 10.7 and 74.5 microM from the Eadie-Hofstee plot. This suggested that there were either two forms of enzyme or two different enzymes participating in ortho hydroxylation of p-nitrophenol in lung microsomes. 5. Lung microsomal p-nitrophenol hydroxylase activity of sheep was reconstituted in the presence of purified lung microsomal cytochrome P-450, NADPH dependent cytochrome P-450 reductase and synthetic lipid, phosphatidylcholine dilauroyl.     

Purification and characterization of the NADPH-cytochrome P-450 (cytochrome c) reductase from higher-plant microsomal fraction.

NADPH-cytochrome P-450 (cytochrome c) reductase (EC 1.6.2.4) was solubilized by detergent from microsomal fraction of wounded Jerusalem-artichoke (Helianthus tuberosus L.) tubers and purified to electrophoretic homogeneity. The purification was achieved by two anion-exchange columns and by affinity chromatography on 2',5'-bisphosphoadenosine-Sepharose 4B. An Mr value of 82,000 was obtained by SDS/polyacrylamide-gel electrophoresis. The purified enzyme exhibited typical flavoprotein redox spectra and contained equimolar quantities of FAD and FMN. The purified enzyme followed Michaelis-Menten kinetics with Km values of 20 microM for NADPH and 6.3 microM for cytochrome c. In contrast, with NADH as substrate this enzyme exhibited biphasic kinetics with Km values ranging from 46 microM to 54 mM. Substrate saturation curves as a function of NADPH at fixed concentration of cytochrome c are compatible with a sequential type of substrate-addition mechanism. The enzyme was able to reconstitute cinnamate 4-hydroxylase activity when associated with partially purified tuber cytochrome P-450 and dilauroyl phosphatidylcholine in the presence of NADPH. Rabbit antibodies directed against plant NADPH-cytochrome c reductase affected only weakly NADH-sustained reduction of cytochrome c, but inhibited strongly NADPH-cytochrome c reductase and NADPH- or NADH-dependent cinnamate hydroxylase activities from Jerusalem-artichoke microsomal fraction.     

Formation of benzoquinol moiety in cornoside by salidroside mono-oxygenase,a cytochrome P450 enzyme,from <Emphasis Type="Italic">Abeliophyllum distichum </Emphasis>cell suspension cultures

A microsomal fraction prepared from Abeliophyllum distichumNakai (Oleaceae) cell suspension cultures oxidized salidroside, a glucoside of 4-hydroxyphenylethyl alcohol, to cornoside possessing a unique benzoquinol ring. The enzyme named salidroside mono-oxygenase required NADPH as the only cofactor, and molecular oxygen. The reaction was strongly inhibited by CO as well as several cytochrome P450 inhibitors, such as cytochrome c and miconazole, indicating the involvement of a cytochrome P450 enzyme. Salidroside mono-oxygenase accepted salidroside as the only substrate, but did not oxidize 4-hydroxyphenylethyl alcohol, the salidroside aglucone, and 4-hydroxybenzoic acid. The optimum pH of the reaction was 7.5, and apparent K(m) values for salidroside and NADPH were 44 micro M and 33 micro M, respectively. The benzoquinol ring formation mechanism is discussed in comparison to the mechanism for ipso substitution of 4-hydroxybenzoate by active oxygen species followed by elimination leading to hydroquinone.     

Taxoid metabolism: Taxoid 14beta-hydroxylase is a cytochrome P450-dependent monooxygenase

The production of the anticancer drug Taxol in Taxus (yew) cell cultures is often accompanied by the formation of side-route polyoxygenated taxoid metabolites bearing a 14beta-hydroxyl group. The recent acquisition of several new semisynthetic taxoid intermediates enabled the screening of a family of Taxus cytochrome P450 cDNA clones for the 14beta-hydroxylase and additional taxoid oxygenases. The candidate cytochrome P450 clones were functionally expressed in yeast and tested by in vivo feeding of radiolabeled 5alpha-acetoxy-10beta-hydroxy taxadiene and 5alpha,13alpha-dihydroxy taxadiene. One clone efficiently and specifically transformed the 5alpha-acetoxy-10beta-ol, but not the 5alpha,13alpha-diol, to a more polar product with the chromatographic properties of a taxoid triol monoacetate, and the identity of this product was confirmed by spectroscopic means as 5alpha-acetoxy-10beta,14beta-dihydroxy taxadiene. Microsome preparation from the transformed yeast allowed characterization of this new hydroxylase, which was shown to resemble other cytochrome P450 taxoid hydroxylases with pH optimum at 7.5 and a K(m) value for the taxoid substrate of about 50 microM. Because Taxol is unsubstituted at C14, the 14beta-hydroxylase cannot reside on the pathway to the target drug but rather appears to be responsible for diversion of the pathway to 14-hydroxy taxoids that are prominent metabolites of Taxus cell cultures. Manipulation of this hydroxylase gene could permit redirection of the pathway to increase flux toward Taxol and could allow the preparation of 13alpha,14beta-hydroxy taxoids as new therapeutic agents.     

NADPH:cytochrome P-450(c) reductase: Biochemical characterization in rat brain and cultured neurons and evolution of activity during development

NADPH:cytochrome P-450 (c) reductase is a microsomal enzyme which is involved in the cytochrome P-450-dependent biotransformation of many exogenous agents as well as of some endogenous molecules. Using cytochromec as a substrate, the kinetic parameters of this enzyme were determined in brain microsomes. The comparison of the NADPH:cytochrome P-450 reductase's V_max values and cytochrome P-450 contents in both fractions, suggests a role of cerebral NADPH:cytochrome P-450 reductase in cytochrome P-450 independent pathways. This is also supported by the different developmental pattern of brain enzyme as compared to the liver enzyme, and by the presence of a relatively high NADPH:cytochrome P-450 reductase activity in immature rat brain and neuronal cultures, while cytochrome P-450 was hardly detectable in these preparations. The enzyme activity was not induced by a phenobarbital chronic treatment neither in the adult brain nor in cultured neurons, suggesting a different regulation of the brain enzyme expression.     

Induction of microsomal aryl hydrocarbon (3,4-benzo(α)pyrene) hydroxylase and cytochrome P-450k in rat kidney cortex: I. Characteristics of the hydroxylase system

Both the rat kidney cortex aryl hydrocarbon hydroxylase activity and cytochrome P-450_K are induced by benzo(α)pyrene treatment. Following a single injection of benzo(α)pyrene, maximal hydroxylase activity and cytochrome P-450_K content occur at 24 hr, returning to control levels within 72–96 hr. Induction of both the enzyme activity and hemoprotein is inhibited by cycloheximide. The enzyme system is localized in the microsomal fraction, has an absolute requirement for NADPH and molecular oxygen, and a pH optimum at 7.4; the induced activity is linear with microsomal protein concentration up to 0.8 mg and with time up to 20 min. Both the hydroxylase activity and cytochrome P-450_K follow the same pattern of inactivation with increasing temperature. The apparent K_m for the induced hydroxylase was 7.7 μm and V was increased fourfold above control value. In the presence of laurate, a substrate for the kidney microsomal cytochrome P-450_K-dependent monooxygenase system, the amount of inhibition of hydroxylase activity corresponded to the level of activity present in untreated kidney cortex microsomes. α-Naphthoflavone (10^?5m), a type I inducer (36) produced a greater inhibitory effect on the induced hydroxylase activity than on the control (55% vs 20%). The presence of cytochrome c or carbon monoxide markedly decreased hydroxylase activity. This evidence in addition to aforementioned characteristics of the enzyme suggests a cytochrome P-450_K-dependent aryl hydroxylase activity which differs from that present in the control rat.     

Phytoalexin synthesis in soybean: purification and reconstitution of cytochrome P450 3,9-dihydroxypterocarpan 6a-hydroxylase and separation from cytochrome P450 cinnamate 4-hydroxylase

Elicitor-challenged soybean (Glycine max) cell cultures were used for detergent solubilization and purification of cytochrome P450 3,9-dihydroxypterocarpan 6a-hydroxylase (D6aH). D6aH was purified to electrophoretic homogeneity from such cells by a five-step procedure. It could be separated from cytochrome P450 cinnamate 4-hydroxylase on hydroxyapatite. This is the first report on separation of two cytochrome P450 enzymes from a higher plant. On sodium dodecyl sulfate polyacrylamide gels D6aH migrated with a Mr about 55,000. For reconstitution experiments soybean NADPH:cytochrome P450 (cytochrome c) reductase was purified to homogeneity. Reconstitution of D6aH in the presence of NADPH was dependent on cytochrome P450 D6aH, the reductase, and lipid. Dilauroylphosphatidylcholine gave higher D6aH activity than soybean lipids (asolectin). The reconstituted D6aH system showed a much higher temperature stability than the microsomal system.     

Purified fusion enzyme between rat cytochrome P4501A1 and yeast NADPH-cytochrome P450 oxidoreductase

A genetically engineered fusion enzyme between rat P4501A1 and yeast P450 reductase in the microsomal fraction of the recombinant yeast AH22/pAFCR1 was purified. The purified enzyme showed a typical CO-difference spectrum of P4501A1 and a single band with an apparent molecular weight of 125,000 on sodium dodecyl sulfate polyacrylamide gel electrophoresis. This agreed with the molecular weight of 131,202 calculated from the amino acid sequence. The purified enzyme showed both 7-ethoxycoumarin o-deethylase activity and horse heart cytochrome c reductase activity in the presence of NADPH. The 7-ethoxycoumarin o-deethylase activity depended on the species of lipid used for the reconstitution of the purified fusion enzyme although the purified enzyme showed the activity without reconstitution. The purified fusion enzyme had the Km value of 26 microM for 7-ethoxycoumarin and the maximal turnover rate of 29 mol product/min/mol enzyme at 30 degrees C.     

Immunochemical studies on the contribution of NADPH cytochrome P-450 reductase to the cytochrome P-450-dependent metabolism of arachidonic acid

We have studied the role of NADPH cytochrome P-450 reductase in the metabolism of arachidonic acid and in two other monooxygenase systems: aryl hydrocarbon hydroxylase and 7-ethoxyresorufin-o-deethylase. Human liver NADPH cytochrome P-450 reductase was purified to homogeneity as evidenced by its migration as a single band on SDS gel electrophoresis, having a molecular weight of 71,000 Da. Rabbits were immunized with the purified enzyme and the resulting antibodies were used to evaluate the involvement of the reductase in cytochrome P-450-dependent arachidonic acid metabolism by bovine corneal epithelial and rabbit renal cortical microsomes. A highly sensitive immunoblotting method was used to identify the presence of NADPH cytochrome P-450 reductase in both tissues. We used these antibodies to demonstrate for the first time the presence of cytochrome c reductase in the cornea. Anti-NADPH cytochrome P-450 reductase IgG, but not anti-heme oxygenase IgG, inhibited the NADPH-dependent arachidonic acid metabolism in both renal and corneal microsomes. The inhibition was dependent on the ratio of IgG to microsomal protein where 50% inhibition of arachidonic acid conversion by cortical microsomes was achieved with a ratio of 1:1. A higher concentration of IgG was needed to achieve the same degree of inhibition in the corneal microsomes. The antibody also inhibited rabbit renal cortical 7-ethoxyresorufin-o-deethylase activity, a cytochrome P-450-dependent enzyme. However, the anti-NADPH cytochrome P-450 reductase IgG was much less effective in inhibiting rabbit cortical aryl hydrocarbon hydroxylase. Thus, the degree of inhibition of monooxygenases by anti-NADPH cytochrome P-450 reductase IgG is variable. However, with respect to arachidonic acid, NADPH cytochrome P-450 reductase appears to be an integral component for the electron transfer to cytochrome P-450 in the oxidation of arachidonic acid.     

Microsomal cytochrome P-450 from neonatal pig testis: two enzymatic activities (17 alpha-hydroxylase and c17,20-lyase) associated with one protein

Studies have been performed to test the hypothesis that cytochrome P-450 from testicular microsomes consists of a single protein with two enzymatic activities (17 alpha-hydroxylase and C17,20-lyase). Three lines of evidence to support the hypothesis were obtained. (1) The enzyme appears to be homogeneous by immunochemical criteria with anti-P-450 IgG (line of identity on immunodiffusion and a single band on immunoelectrophoresis), by demonstration of a single NH2-terminal amino acid (methionine) and the finding of 16 single amino acids at the NH2 terminus. (2) Optima for pH and temperature are the same for both enzymatic activities (pH 7.25 and 37 degrees C), and temperatures between 30 and 44 degrees C decreased both activities in such a way that the ratio of hydroxylase to lyase was the same at all temperatures tested. (3) A variety of inhibitors affect both activities to the same extent: Ki values for two competitive inhibitors (SU 8000, 0.04 microM; SU 10603, 0.3 microM) are the same for hydroxylase and lyase; partition coefficients for inhibition by carbon monoxide are similar for hydroxylase and lyase (20 +/- 2 and 27 +/- 3); anti-P-450 (serum and IgG) causes inhibition of both activities to the same extent, and the same is true of a variety of less specific inhibitors. It is concluded that a single heme protein (cytochrome P-450) from microsomes of neonatal pig testis catalyzes two reactions (hydroxylase and lyase) which are sequential steps in the synthesis of androgens by the testis leading to conversion of C21 precursors to C19 steroid hormones.     

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

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.     

Physicochemical properties of reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase from bovine adrenocortical microsomes.

Adrenocortical NADPH-cytochrome P-450 reductase (EC. 1.6.2.4) was purified from bovine adrenocortical microsomes by detergent solubilization and affinity chromatography. The purified cytochrome P-450 reductase was a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, being electrophoretically homogeneous and pure. The cytochrome P-450 reductase was optically a typical flavoprotein. The absorption peaks were at 274, 380 and 45 nm with shoulders at 290, 360 and 480 nm. The NADPH-cytochrome P-450 reductase was capable of reconstituting the 21-hydroxylase activity of 17 alpha-hydroxyprogesterone in the presence of cytochrome P-45021 of adrenocortical microsomes. The specific activity of the 21-hydroxylase of 17 alpha-hydroxyprogesterone in the reconstituted system using the excess concentration of the cytochrome P-450 reductase, was 15.8 nmol/min per nmol of cytochrome P-45021 at 37 degrees C. The NADPH-cytochrome P-450 reductase, like hepatic microsomal NADPH-cytochrome P-450 reductase, could directly reduce the cytochrome P-45021. The physicochemical properties of the NADPH-cytochrome P-450 reductase were investigated. Its molecular weight was estimated to be 80 000 +/- 1000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical ultracentrifugation. The cytochrome P-450 reductase contained 1 mol each FAD and FMN as coenzymes. Iron, manganese, molybdenum and copper were not detected. The Km values of NADPH and NADH for the NADPH-cytochrome c reductase activity and those of cytochrome c for the activity of NADPH-cytochrome P-450 reductase were determined kinetically. They were 5.3 microM for NADPH, 1.1 mM for NADH, and 9-24 microM for cytochrome c. Chemical modification of the amino acid residues showed that a histidyl and cysteinyl residue are essential for the binding site of NADPH of NADPH-cytochrome P-450 reductase.     

Cytochrome P-450 dependent metabolism of testosterone in rat skin

The incubation of microsomes of whole skin, dermis and epidermis with 14C testosterone in the presence of NADPH resulted in the formation of 6 beta-, 7 alpha- and 16 alpha-testosterone. Maximum enzyme activity occurred in epidermal microsomes followed by dermis and whole skin. Epidermal testosterone hydroxylase activity required NADPH and oxygen and was found to be inhibited by SKF 525A and metyrapone. Our data strongly suggest that testosterone is metabolized by the cytochrome P-450 dependent monooxygenase in skin and provides the first evidence for an endogenous substrate for cytochrome P-450 in this tissue. The formation of several hydroxylated products further suggests the existence of multiple isozymes of cytochrome P-450 in rat skin. These studies provide additional evidence that target tissues may modulate their hormone levels by enzyme pathways that are locally regulated.     

Kinetic and structural properties of biocatalytically active sheep lung microsomal NADPH-cytochrome c reductase

NADPH-cytochrome c reductase was purified to electrophoretic homogeneity from detergent solubilized sheep lung microsomes. The specific activity of the purified enzyme ranged from 56 to 67 mumol cytochrome c reduced/min/mg protein and the yield was 48-52% of the initial activity in lung microsomes. The reductase had Mr of 78,000 and contained 1 mol each of FAD and FMN. Km values obtained in 0.3 M phosphate buffer, pH 7.8 at 37 degrees C for NADPH and cytochrome c were 11.1 +/- 0.70 microM and 20.0 +/- 2.15 microM. Lung reductase was inhibited by its substrate, cytochrome c when its concentration was above 160 microM. The lung reductase exhibited a ping-pong type kinetic mechanism for NADPH mediated cytochrome c reduction. Purified lung reductase was biocatalytically active in supporting benzo(a)pyrene hydroxylation reaction when coupled with lung cytochrome P-450 and lipid.