NADPH-cytochrome P450 Oxidoreductase Research Articles

Purification and characterization of NADPH-cytochrome P-450 reductase from rat epidermis.

NADPH-cytochrome P-450 oxidoreductase (P-450 red) transfers reducing equivalents from NADPH to cytochrome P-450 (P-450) in the monooxygenase system. Detergent solubilized proteins from the membrane fraction of neonatal rat epidermis were purified by 2',5'-ADP-agarose affinity column chromatography. The purified protein showed an apparent homogeneity on sodium dodecylsulfate-polyacrylamide gel electrophoresis and molecular weight was estimated to be 78 kDa. NADPH-cytochrome c reductase activity increased by 95-fold in the purified enzyme. Epidermal P-450 red in vitro reconstituted benzo(a)pyrene hydroxylase activity in a dose dependent manner with P-450 purified from either rat liver or epidermis. Western blot analysis demonstrated that epidermal P-450 red immunologically cross reacts to liver P-450 red. Immunohistochemical staining showed that the enzyme was predominantly localized in the epidermis. The intensity of immunohistochemical staining of rat skin sections and tissue distribution did not change in the skin treated with beta-naphthoflavone, which results in a substantial increase in P-450 1A1 activity. Quantitative assessment of P-450 red in treated and untreated epidermis also showed no change. These findings indicate that constitutive P-450 red, fully capable of supporting P-450, exists in rat epidermis, and can function in metabolism of endogenous and exogenous compounds.

Inhibition of cytochrome P450 reductase by the diphenyliodonium cation. Kinetic analysis and covalent modifications.

Diphenyliodonium has been shown to be an irreversible, time-dependent inhibitor of NADPH cytochrome P450 oxidoreductase (EC 1.6.2.4) with the Ki for diphenyliodonium chloride being 2.8 mM. Kinetic studies have indicated that diphenyliodonium interacts with the reduced enzyme and NADPH is essential for inactivation to take place. Cytochrome c acts as a competitive substrate. The use of radiolabeled diphenyliodonium has enabled two sites of covalent modification to be identified. Isolation of radiolabeled cofactor followed by mass spectrometry has shown that a phenyl group is added to FMN while the FMN is effectively trapped in the reduced state. Trypsin digestion of S-carboxymethylated P450 reductase after inhibition with radiolabeled inhibitor shows covalent modification of the protein. Purification of a single radiolabelled peptide followed by automated Edman degradation has enabled identification of the second site of covalent attachment as Trp 419.

Formation of 19(S)-, 19(R)-, and 18(R)-hydroxyeicosatetraenoic acids by alcohol-inducible cytochrome P450 2E1

When reconstituted with cytochrome b5 and NADPH cytochrome P450 oxidoreductase, cytochrome P450 2E1 metabolized lauric, stearic, oleic, linoleic, linolenic, and arachidonic acid to multiple metabolites. Two major metabolites, accounting for 78% of the total metabolism, were produced with arachidonic acid. The Vmax for total metabolite formation from arachidonic acid was 5 nmol/min/nmol P450 with an apparent Km of 62 microM. Gas chromatography-mass spectrometry analysis identified the two major metabolites as monohydroxylated eicosatetraenoic acids (HETEs). The major HETE was 19-hydroxyeicosatetraenoic acid (19-HETE) and comprised 46% of the total metabolite produced. The second metabolite was the omega-2 hydroxylated metabolite (18-HETE) and comprised 32% of the total product formed. Chiral analysis demonstrated that 19-HETE was 70% 19(S)-HETE and 30% 19(R)-HETE. In contrast, 18-HETE was essentially 100% R isomer. Approximately 18% of the total metabolite produced from arachidonic acid coeluted with epoxyeicosatrienoic acid (EET) standards. The EET metabolites were 56.4% 14,15-EET and 43.6% as a mixture of 11,12-EET and 8,9-EET. 5,6-EET was not detected. Anti-P450 2E1 IgG inhibited arachidonic acid metabolism by renal and hepatic microsomes prepared from acetone-treated rabbits. With renal cortex microsomes, the formation of 18-HETE and 19-HETE was inhibited 67 and 25%, respectively, by the antibody. Liver microsomal formation of 18-HETE was inhibited by 87% and 19-HETE by 70%. Thus, under conditions where cytochrome P450 2E1 is induced, the enzyme could contribute significantly to the formation of the omega-1 and omega-2 hydroxylated metabolites of arachidonic acid.

Regulation of Phenobarbital-Inducible Cytochrome-P450 2B1/2 mRNA by Lovastatin and Oxysterols in Primary Cultures of Adult Rat Hepatocytes

Lovastatin (LOVA) is a potent inhibitor of 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase widely used in clinical practice. We treated primary cultures of adult rat hepatocytes, maintained in a minimal, serum-free medium on Matrigel, a reconstituted basement membrane, with this drug, and found that the amounts of P450 2B2 mRNA detected on Northern blots were increased at the same doses (10−5 to 3 × 10−5 M) required for induction of HMG-CoA reductase mRNA, a gene known to be under oxysterol regulatory control. LOVA treatment produced selective effects increasing also the mRNA levels for P450s 2C6, 2C7, 3A1, and 4A1 but not for 1A1, 2A1/2, or NADPH-cytochrome P450 oxidoreductase. LOVA treatment increased the induction of 2B1/2 mRNA in cells cotreated with either phenobarbital (PB; 10−4 M) or clotrimazole (CTZ; 10−5 M), or of 3A1 mRNA in cells cotreated with PB (2 × 10−3 M), but not dexamethasone (10−5 M). LOVA treatment did not potentiate the induction of 1A1 or 4A1 mRNA in cells cotreated with β-naphthoflavone (10−5 M) or ciprofibrate (10−4 M), respectively. In contrast to the potentiation of 2B1/2 mRNA induction produced by treatments with LOVA in combination with PB or CTZ, cotreatment of hepatocytes with PB and CTZ did not result in increased induction relative to that seen in cells treated with either agent alone. Treatment of hepatocyte cultures with either mevalonate (3 × 10−4 to 3 × 10−3 M), the immediate product of HMG-CoA reductase, or 25-hydroxycholesterol (10−6 to 10−5 M), a model oxysterol, resulted in dose-dependent suppression of 2B1/2 mRNA induction in cells treated with PB-like inducers. Taken together, our results demonstrate that LOVA is a unique inducer of P450 mRNA in cultured rat hepatocytes and implicate oxysterols as potential intracellular modulators of 2B1/2 induction. We conclude that endogenous metabolic factors including those related to cholesterol biosynthesis are critical in induction of liver cytochromes P450 2B1 and 2B2 by PB and "PB-like" agents.

Enzyme-substrate binding interactions of NADPH-cytochrome P-450 oxidoreductase characterized with pH and alternate substrate/inhibitor studies.

The pH dependence of the kinetic parameters for the reaction catalyzed by NADPH-cytochrome P-450 oxidoreductase (P-450R) has been determined, using various substrates and inhibitors. All Vmax and (V/K) profiles show pKas of 6.2-7.3, for an acidic group that is preferentially unprotonated for catalysis, and of 8.1-9.6, for a basic group that is preferentially protonated for catalysis. The presence of the wrong ionization state for both of these groups is tolerated more at lower ionic strength (300 mM) than at higher ionic strength (850 mM). Ionization of the basic group has a more pronounced effect on binding of substrate (cytochrome c or dichloroindophenol) than on catalysis, since ionization has only a 2-fold effect on Vmax with cytochrome c, and only a 5-fold effect on Vmax with dichloroindophenol, while (V/K) for both substrates continues to drop at high pH with no sign of reaching a plateau. Therefore, this basic group affects predominantly substrate binding and, to a lesser extent, catalysis. It is most likely located on the surface of the protein at the cytochrome c/dichloroindophenol binding site, near the FMN prosthetic group. The NADP+ pKi profile shows a pKa of 5.95 for the 2'-phosphate of NADP+, which is bound to P-450R as the dianion, and a pKa of 9.53 for an enzyme group that must be protonated in order to bind NADP+.(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction with arginine 597 of NADPH-cytochrome P-450 oxidoreductase is a primary source of the uniform binding energy used to discriminate between NADPH and NADH.

Site-directed mutagenesis has been used in conjunction with pH and alternate substrate/inhibitor studies to characterize the interactions between NADPH-cytochrome P-450 oxidoreductase (P-450R) and the 2'-phosphate of NADP(H) that provide P-450R with its strong nicotinamide nucleotide specificity. It is known that the 2'-phosphate of NADP(H) is bound to P-450R as the dianion and that interactions between it and residues on P-450R provide 5 kcal/mol of essentially uniform binding energy (preceding paper in this issue). In order to probe these interactions further, Arg597 of P-450R, which is homologous to Arg235 of ferredoxin-NADP+ reductase that forms a salt bridge with the 2'-phosphate of 2'-phospho-AMP in the crystal structure of that complex [Karplus, P. A., Daniels, M. J., & Herriott, J. R. (1991) Science 251, 60], was mutated to methionine. The mutant protein, P-450R (R597M), does not appear to have a grossly perturbed tertiary structure on the basis of the observation of similar 31P-NMR chemical shifts for FAD (pyrophosphate) bound to it and wild-type (WT) P-450R, although it is more unstable to urea denaturation. P-450R (R597M) has a Km for NADPH that is 150 times that of P-450R (WT) and a Ki for NADP+ that is 240 times that of P-450R (WT). In contrast, the R597M mutation has only a modest effect on the Km for NADH (0.8 WT) and the Ki for NAD+ (2.9 WT), indicating that Arg597 must have been interacting specifically with the 2'-phosphate of NADP(H). The R597M mutation has relatively little effect on kcat for NADPH (1.2 WT) or NADH (0.6 WT), indicating that the mutation is affecting ground and transition states to essentially the same degree, by removing 3 kcal/mol of uniform binding energy. The NADP+ pKi profile for P-450R (R597M) shows a pKa of 5.78 for the 2'-phosphate of NADP+, which is bound to P-450R (R597M) as the dianion, but the pKa of 9.5 for the preferentially protonated enzymic group observed in the P-450R (WT) profile is no longer present. It is argued then that the 2'-phosphate binding pocket of P-450R (WT) has a high positive charge density (> + 2) and that Arg597, which is in this binding pocket, has a highly perturbed pKa of 9.5. Finally, a general theoretical treatment of the thermodynamic consequences of individual and combined perturbations to complementary interacting groups on enzyme and substrate is presented (see Appendix).(ABSTRACT TRUNCATED AT 400 WORDS)

Role of acidic residues in the interaction of Nadphcytochrome P450 Oxidoreductase with substrates

Role of acidic residues in the interaction of Nadphcytochrome P450 Oxidoreductase with substrates

Expression of a catalytically active human cytochrome P-4502E1 in Escherichia coli

The cDNA coding for human cytochrome P-4502E1 has been expressed in Escherichia coli by placing it under the control of the isopropylthiogalactoside inducible trc promoter. Production of P4502E1 was demonstrated by immunoblots and by catalytic activity with dimethylnitrosamine as substrate. Modifications which favor expression in E. coli were made within the first seven codons. This resulted in approx. a 2- to 2.5-fold increase in P4502E1 isolated from the bacterial membranes as detected by immunoblots and catalytic activity. CO-reduced difference spectra of the modified P4502E1 revealed a peak at 452 nm, which is characteristic of hepatic P4502E1, and a molecular weight of 54 kDa. A partially purified preparation of recombinant P450 protein was active with dimethylnitrosamine, a substrate specific for this isozyme, when reconstituted with purified rat liver NADPH-cytochrome P-450 oxidoreductase. This activity was enhanced in the presence of cytochrome b 5 and inhibited by the addition of antibody to the P4502E1 purified from pyrazole-treated rats. E. coli were capable of oxidizing p-nitrophenol when transformed with vector containing the human P4502E1 cDNA but not with vector alone. This in vivo metabolism of p-nitrophenol was increased 2-fold when the modified P4502E1 cDNA was used, which corresponds to the increase in P4502E1 content. Expression of human P4502E1 in E. coli appears to be an attractive system for producing large amounts of this isozyme, and for studies on the toxicological properties and structure-function relationship of the human P4502E1.

Molecular cloning and sequence analysis of mouse NADPH-cytochrome P-450 oxidoreductase

A cDNA clone coding for cytochrome P-450 oxidoreductase was isolated from a mouse liver cDNA library. The cDNA, MSr2, contained a complete coding region of 678 amino acids. The amino acid sequence of the mouse cytochrome P-450 oxidoreductase showed approx. 90% identities with those of rat, human, rabbit, pig enzymes indicating conservation of primary structure of the enzyme during evolutionary divergence of species. The high conservation of acidic residues of the enzyme sustained the importance of them to maintain its function.

Geometric relationship between the nicotinamide and isoalloxazine rings in NADPH-cytochrome P-450 oxidoreductase: implications for the classification of evolutionarily and functionally related flavoproteins.

The stereospecificity of hydride abstraction from NADPH and the conformation of the nicotinamide ring around the glycosidic bond have been determined for the flavoprotein NADPH-cytochrome P-450 oxidoreductase (P-450R). The A-side (pro-R) hydrogen is abstracted from NADPH, and the nicotinamide ring is in the anti conformation. These results are consistent with the apparently strong correlation between A-side stereospecificity and anti conformation and between B-side stereospecificity and syn conformation [You, K. (1985) CRC Crit. Rev. Biochem. 17, 313]. This correlation reveals how the flavin and nicotinamide rings are oriented relative to each other. In P-450R, the flavin is then "on top of" (on the exo side of) the nicotinamide ring. In another flavoprotein dehydrogenase, glutathione reductase, which is a B-side/anti enzyme [Pai, E. F., & Schulz, G. E. (1983) J. Biol. Chem. 258, 1752], the flavin is "underneath" (on the endo side of) the nicotinamide ring. We argue that all enzymes that are evolutionarily related to these two flavoproteins should have their respective overall configurations. The overall configuration is defined by the following five properties: (1) relative orientation of the isoalloxazine and nicotinamide rings, (2) stereospecificity of hydride transfer to/from the nicotinamide ring, (3) conformation of the nicotinamide ring around the glycosidic bond, (4) stereospecificity of hydride transfer to/from the flavin, and (5) conformation of the flavin around its N5-N10 axis. There are only eight possible overall configurations, and a knowledge of only three of the five properties is needed to determine which one is present (as long as the combination of properties is not 1, 2, 3 or 1, 4, 5).(ABSTRACT TRUNCATED AT 250 WORDS)

Expression, purification, and properties of the flavoprotein domain of cytochrome P-450BM-3. Evidence for the importance of the amino-terminal region for FMN binding.

Comparison of the amino acid sequences of several microsomal cytochrome P-450 reductases to the flavoprotein domain (BMR) of cytochrome P-450BM-3 has revealed that this class of flavoproteins contains evolutionarily conserved regions that are important for their interaction with nucleotide substrates and cofactors. In order to understand the properties of BMR, the region encoding this protein, beginning at residue Lys-472 of cytochrome P-450BM-3, was subcloned and expressed in Escherichia coli. The recombinant protein (more than 50% of host-soluble proteins) was purified to homogeneity using conventional purification procedures. BMR (Mr 66,000) showed typical flavoenzyme absorbance spectra, contained FAD and FMN in a stoichiometry of 1:1, and catalyzed reduction of several artificial electron acceptors with rates comparable to those of the microsomal NADPH-cytochrome P-450 oxidoreductase. Limited trypsinolysis of BMR, under non-denaturing conditions, revealed that the protease removed the NH2-terminal 122 residues. This region was postulated to contain amino acids that are important for FMN binding (Porter, T. D. (1991) Trends Biochem. Sci. 16, 154-158). Consistent with this hypothesis, the major tryptic product of BMR (BMR-52, Mr 52,000) contained only FAD, in an equimolar ratio to the protein. Also, like the FMN-depleted microsomal NADPH-cytochrome P-450 oxidoreductase (Kurzban, G. P., Howarth, J., Palmer, G., and Strobel, H. W. (1990) J. Biol. Chem. 265, 12272-12279), BMR-52 was active for only catalyzing ferricyanide reduction. These data provide strong experimental evidence for a discrete multidomain structure of BMR, as proposed for the membrane-bound reductases, with an amino-terminal FMN binding region and carboxyl-terminal FAD- and NADPH binding regions. Thus, BMR strongly resembles the microsomal cytochrome P-450 reductase and offers an opportunity to better understand the structure-function relationships of this class of flavoproteins.

NADPH-cytochrome P-450 oxidoreductase. The role of cysteine 566 in catalysis and cofactor binding.

Site-directed mutagenesis was employed to investigate the role of Cys566 in the catalytic mechanism of rat liver NADPH-cytochrome P-450 oxidoreductase. Rat NADPH-cytochrome P-450 oxidoreductase and mutants containing either alanine or serine at position 566 were expressed in Escherichia coli and purified to homogeneity. Substitution of alanine at position 566 had no effect on enzymatic activity with the acceptors cytochrome c and ferricyanide but did increase trans-hydrogenase activity with 3-acetylpyridine adenine dinucleotide phosphate by 79%. The Km for NADPH was increased 2.5-fold, and the NADP+ KI was increased 4.8-fold compared with that found for the wild-type enzyme. The conservative substitution, Ser566, produced a 50% decrease in cytochrome c reductase activity whereas activity with ferricyanide was decreased 57%, and 3-acetylpyridine adenine dinucleotide phosphate activity was unaffected. The NADPH Km was increased 4.6-fold, and the NADP+ KI increased 7.6-fold. The dependence of cytochrome c reductase activity on the KCl concentration was markedly altered by the Cys566 substitutions. Maximum activity for the wild-type enzyme was observed at approximately 0.18 M KCl whereas maximum activity for the mutant enzymes was observed between 0.04 and 0.09 M KCl. The pH dependence of cytochrome c reductase activity, cytochrome c Km, and flavin content were unaffected by these substitutions. These results demonstrate that Cys566 is not essential for activity of rat liver NADPH-cytochrome P-450 oxidoreductase although the cysteine side chain does affect the interaction of NADPH with the enzyme.

Transcriptional regulation and localization of the tissue-specific induction of epoxide hydrolase by lead acetate in rat kidney.

Intraperitoneal administration of lead acetate to male Sprague-Dawley rats resulted in the tissue-specific transcriptional activation of the microsomal epoxide hydrolase gene in kidney. This response was followed by a 15-fold increase in the level of kidney epoxide hydrolase mRNA, while no change in mRNA level was noted in liver. Treated animals also showed no increase in mRNA levels for NADPH-cytochrome P-450 oxidoreductase, cytochrome P-450b/e, cytochrome P-450PCN (where PCN is pregnenolone 16 alpha-carbonitrile), or serum albumin in either kidney or liver. Immunoquantitation of the enzyme revealed a 16-fold increase in kidney upon treatment with lead acetate, but significant changes in epoxide hydrolase levels were not noted in liver, heart, spleen, lung, small intestine, or testis. The enzymatic activity for liver and kidney paralleled the immunochemical results; however, the activity increase in kidney was only one-third of the increase noted for total enzyme protein. Immunohistochemical analysis of epoxide hydrolase protein in sections of rat kidney demonstrated that in lead acetate-treated animals there was a marked increase in staining of the cytoplasm of the proximal tubular cells in the outer cortex as compared with kidneys from control animals. In contrast, considerable protein was also localized to collecting ducts, but no change was evident in the content of the epoxide hydrolase gene product in these structures in control and lead acetate-treated animals. Immunohistochemical differences were not noted between livers from control and lead-treated animals. Furthermore, the staining patterns for NADPH-cytochrome P-450 oxidoreductase were the same for control and treated animals in both kidney and liver. Quantitative measurements of lead uptake by various rat tissues showed liver, spleen, and small intestine reaching a maximum of approximately 12,000 ng of lead/g of dry tissue at 8, 8, and 16 h, respectively, while kidney, lung, and testis peaked (approximately 3,000 ng of lead/g of dry tissue) at 16, 16, and 12 h, respectively.

NADPH-cytochrome P-450 oxidoreductase gene organization correlates with structural domains of the protein

cDNA clones to rat liver NADPH-cytochrome P-450 oxidoreductase were used to isolate genomic clones from a Wistar-Furth inbred rat genomic DNA library. Fifteen exons containing the coding region and 3'-nontranslated segment of the P-450 reductase gene were identified, spanning 20 kilobases of DNA contained in 3 lambda-Charon 35 clones. The organization of this single copy gene reveals a general correspondence between exons and structural domains of the protein, with the segment responsible for anchoring the reductase to the microsomal membrane and several segments involved in FMN, FAD, and NADPH binding encoded by discrete exons.

Isolation and characterization of the alkane-inducible NADPH-cytochrome P-450 oxidoreductase gene from Candida tropicalis. Identification of invariant residues within similar amino acid sequences of divergent flavoproteins.

The gene coding for the Candida tropicalis NADPH-cytochrome P-450 oxidoreductase (CPR, NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4) was isolated by immunoscreening of a C. tropicalis lambda gt11 expression library and colony hybridization of a C. tropicalis genomic library. The C. tropicalis CPR gene produces a 2.35-kilobase mRNA transcript, levels of which were shown to be increased 16-fold in cells grown on tetradecane relative to cells grown on glucose as the sole carbon source. A 3-kilobase DNA fragment was sequenced, including 554 and 397 base pairs of 5'- and 3'-noncoding sequence, respectively. A single open reading frame of 2040 base pairs was identified and predicts a 76,683-Da polypeptide of 680 amino acid residues. The deduced C. tropicalis CPR amino acid sequence was compared with each of the CPR sequences reported from other organisms and invariant residues were identified. Multiple pairwise alignments of divergent members of protein families, previously recognized for their sequence similarities in their respective binding domains for FMN, FAD, and NADPH, have allowed identification of a subset of these invariant residues. From these analyses we infer the importance of 25 of the 680 amino acid residues.

Immunochemical and molecular biological studies on human placental cigarette smoke-inducible cytochrome P-450-dependent monooxygenase activities

The induction of specific forms of cytochrome P-450 and P-450-associated xenobiotic-metabolizing monooxygenase activities by maternal cigarette smoking was characterized in human placenta employing polyclonal and monoclonal antibodies and recombinant DNA probes. The anti-BNF-B 2 (prepared against rat liver P-450 induced by beta-naphthoflavone) inhibited about 60 per cent of aryl hydrocarbon hydroxylase (AHH) and 7-ethoxyresorufin O-deethylase activities (ERDE) in placental tissues from smoking mothers, whereas the anti-PB-B 2 (to phenobarbital-induced rat liver P-450) was without significant inhibitory effect. Inhibition of 7-ethoxycoumarin O-deethylase (ECDE) by the anti-BNF-B 2 was dependent on maternal smoking: the enzyme from non-smokers was not significantly inhibited, whereas the enzyme from smokers was variably inhibited by 15–60 per cent. The monoclonal antibodies towards the major 3-methylcholanthrene-inducible and phenobarbital-inducible rat liver P-450s (Mab 1-7-1 and 2-66-3, respectively) behaved similarly, except the inhibition was somewhat stronger if present. Antibody raised against rat liver NADPH-cytochrome P-450 oxido-reductase did not inhibit any activity studied. In immunoblotting experiments, the anti-reductase recognized the protein in human placental microsomes. However, neither anti-BNF-B 2, anti-PB-B 2 or Mab 1-7-1 or Mab 2-66-3 detected any proteins in human placental microsomes, regardless of smoking status. Northern blot hybridization analysis of placental RNA samples showed that only P-450IA1 mRNA existed in the placentas of smoking mothers with detectable ERDE activity. Despite the discrepancy between protein blotting and immunoinhibition data all other findings support the conclusion that maternal cigarette smoking induces the expression of CYPIA1 gene (and not CYPIA2), resulting in an increased synthesis of P-450IA1 protein and increased AHH, ERDE and ECDE activities in human placenta.

Functional Expression of the Alkane-Inducible Monooxygenase System of the Yeast: Candida tropicalis IN Saccharomyces cerevisiae

The genes for the alkane-inducible monooxygenase system of the yeast Candida tropicalis, namely a cytochrome P450alk (P450alk) and a NADPH cytochrome P450 oxidoreductase (NCPR) gene, were transferred in Saccharomyces cerevisiae. The P450alk gene was expressed in this host with the help of the yeast alcohol dehydrogenase I (ADHI) promoter and terminator, whereas the NCPR gene could be expressed with its own structural elements. The presence of P450alk in S. cerevisiae microsomal fractions resulted in a new acquired lauric acid terminal hydroxylation activity. Moreover, the same activity, coupled with the appearance of 12-hydroxylauric acid derivatives, could be obtained by the addition of lauric acid to intact cells expressing P450alk. The coordinate expression of the P450alk and NCPR genes in S. cerevisiae elevated the turnover rate of the P450alk monooxygenase activity about 2-fold.

Characterization of the Flavoprotein Moieties of NADPH-Sulfite Reductase from Salmonella typhimurium and Escherichia coli: Physico-chemical and catalytic properties, amino acid sequence deduced from dna sequence of cysJ, and comparison with nadph-cytochrome P-450 reductase

Abstract NADPH-sulfite reductase flavoprotein (SiR-FP) was purified from a Salmonella typhimurium cysG strain that does not synthesize the hemoprotein component of the sulfite reductase holoenzyme. cysJ, which codes for SiR-FP, was cloned from S. typhimurium LT7 and Escherichia coli B, and both genes were sequenced. Physicochemical analyses and deduced amino acid sequences indicate that SiR-FP is an octamer of identical 66-kDa peptides and contains 4 FAD and 4 FMN per octamer. Potentiometric titrations of SiR holoenzyme, SiR-FP, and FMN-depleted SiR-FP yielded the following redox potentials for the prosthetic groups at pH 7.7: E'1 (FMNH./FMN) = -152 mV; E'2 (FMNH2/FMNH.) = -327 mV; E'3 (FADH./FAD) = -382 mV; E'4 (FADH2/FADH.) = -322 mV. Microcoulometric titration of SiR-FP at 25 degrees C yielded data which were in full agreement with these potentials. Spectroscopic and catalytic studies of native SiR-FP and of SiR-FP depleted of FMN support the following electron flow sequence: NADPH----FAD----FMN. FMN can then contribute electrons to the hemoprotein component of sulfite reductase, as well as to cytochrome c and various diaphorase acceptors. The FMN is postulated to cycle between the FMNH2 and FMNH. oxidation states during catalysis; in this sense SiR-FP shares a catalytic mechanism with NADPH-cytochrome P-450 oxidoreductase. SiR-FP domains involved in binding FMN, FAD, and NADPH are proposed from amino acid sequence homologies with Desulfovibrio vulgaris flavodoxin (Dubourdieu, M., and Fox, J.L. (1977) J. Biol. Chem. 252, 1453-1463) and spinach ferredoxin-NADP+ oxidoreductase (Karplus, P.A., Walsh, K.A., and Herriott, J. R. (1984) Biochemistry 23, 6576-6583). Comparison of the deduced amino acid sequences of SiR-FP and NADPH-cytochrome P-450 oxidoreductase (Porter, T. D., and Kasper, C.B. (1985) Proc. Natl. Acad. Sci. U. S.A. 82, 973-977) also showed identities that suggest these two proteins are descended from a common precursor, which contained binding regions for both FMN and FAD.

Structural analysis of the FMN binding domain of NADPH-cytochrome P-450 oxidoreductase by site-directed mutagenesis

Comparison of the amino acid sequence of rat liver NADPH-cytochrome P-450 oxidoreductase with that of flavoproteins of known three-dimensional structure suggested that residues Tyr-140 and Tyr-178 are involved in binding of FMN to the protein. To test this hypothesis, NADPH-cytochrome P-450 oxidoreductase was expressed in Escherichia coli using the expression-secretion vector pIN-III-ompA3, and site-directed mutagenesis was employed to selectively alter these residues and demonstrate that they are major determinants of the FMN-binding site. Bacterial expression produced a membrane-bound 80-kDa protein containing 1 mol each of FMN and FAD per mol of enzyme, which reduced cytochrome c at a rate of 51.5 mumol/min/mg of protein and had absorption spectra and kinetic properties very similar to those of the rat liver enzyme. Replacement of Tyr-178 with aspartate abolished FMN binding and cytochrome c reductase activity. Incubation with FMN increased catalytic activity to a maximum of 8.6 mumol/min/mg of protein. Replacement of Tyr-140 with aspartate did not eliminate FMN binding, but reduced cytochrome c reductase activity about 5-fold, suggesting that FMN may be bound in a conformation which does not permit efficient electron transfer. Substitution of phenylalanine at either position 140 or 178 had no effect on FMN content or catalytic activity. The FAD level in the Asp-178 mutant was also decreased, suggesting that FAD binding is dependent upon FMN; FAD incorporation may occur co-translationally and require prior formation of an intact FMN domain.

Quantitation of mRNAs specific for the mixed-function oxidase system in rat liver and extrahepatic tissues during development

Evaluation of ontogenetic expression of the cytochrome P450PCN and cytochrome P450b gene families as well as the NADPH-cytochrome P450 oxidoreductase and epoxide hydrolase genes in Holtzmann rats showed that basal levels of mRNAs encoding these enzymes could be detected in most tissues. Distinct developmental patterns of mRNA expression are evident for these four proteins in liver and extrahepatic tissues. Levels of cytochrome P450b-like mRNA were comparable in adult lung and liver, while cytochrome P450PCN-homologous mRNA exhibited low levels in lung and approximately 100-fold higher levels in liver. Cytochrome P450PCN-homologous mRNA also reached substantial levels in adult intestine, and was also present in placenta, where it increased approximately 4-fold 24 h before birth. Epoxide hydrolase mRNA was demonstrated to be highest in liver followed by kidney, lung, and intestine but was extremely low in brain. NADPH-cytochrome P450 oxidoreductase mRNA in kidney, lung, prostate, adrenal, and intestine exhibited levels comparable to that found in liver; however, the pattern of expression for oxidoreductase mRNA was unique in that levels declined at maturity in liver, kidney, and intestine but not in lung and brain. Development of mixedfunction oxidase and epoxide hydrolase activities in liver was distinct from that in other tissues in that mRNAs for all four proteins rose dramatically after parturition. Testis from immature males demonstrated low levels of all the mRNAs assayed, which ranged from 20% (oxidoreductase) to less than 1% (cytochrome P450PCN and epoxide hydrolase) of the levels found in liver.