A cytochrome P450 and a ferredoxin isolated from Mycobacterium sp. strain HE5 after growth on morpholine

A cytochrome P450 and an iron-sulfur protein, whose expression was specifically induced during growth of Mycobacterium sp. strain HE5 on morpholine as the sole source of carbon, nitrogen, and energy were purified to apparent homogeneity. Due to the lack of enzymatic activity, carbon monoxide difference spectra and determination of the acid-labile sulfur, respectively, were used to detect the proteins during purification. The cytochrome P450, designated P450mor, was characterized as a monomer with an apparent molecular mass of 44.7 kDa. The amino acid sequence of an internal peptide comprising 19 amino acids was identical to the sequence derived from a gene encoding a cytochrome P450 from Mycobacterium smegmatis mc(2)155 suggested to be involved in the utilization of piperidine and pyrrolidine. The iron-sulfur protein was characterized as a ferredoxin exhibiting a molecular mass of 6.8 kDa and named Fdmor. An identity of 48-77% was obtained for the 30 N-terminal amino acids of Fdmor and the corresponding sequences of different 3Fe-4S-ferredoxins known to be involved in P450-dependent reactions. From these data we concluded that growth of Mycobacterium sp. strain HE5 on morpholine led to the expression of a cytochrome P450-dependent monooxygenase system composed of at least two different proteins.     

Site-specific mutations in human ferredoxin that affect binding to ferredoxin reductase and cytochrome P450scc

Ferredoxins found in animal mitochondria function in electron transfer from NADPH-dependent ferredoxin reductase (Fd-reductase) to cytochrome P450 enzymes. To identify residues involved in binding of human ferredoxin to its electron transfer partners, neutral amino acids were introduced in a highly conserved acidic region (positions 68-86) by site-directed mutagenesis of the cDNA. Mutant ferredoxins were produced in Escherichia coli, and separate assays were used to determine the effect of substitutions on the capacity of each mutant to bind to Fd-reductase and cytochrome P450scc and to participate in the cholesterol side chain cleavage reaction. Replacements at several positions (mutants D68A, E74Q, and D86A) did not significantly affect activity, suggesting that acidic residues at these positions are not required for binding or electron transfer interactions. In contrast, substitutions at positions 76 and 79 (D76N and D79A) caused dramatic decreases in activity and in the affinity of ferredoxin for both Fd-reductase and P450scc; this suggests that the binding sites on ferredoxin for its redox partners overlap. Other substitutions (mutants D72A, D72N, E73A, E73Q, and D79N), however, caused differential effects on binding to Fd-reductase and P450scc, suggesting that the interaction sites are not identical. We propose a model in which Fd-reductase and P450scc share a requirement for ferredoxin residues Asp-76 and Asp-79 but have other determinants that differ and play an important role in binding. This model is consistent with the hypothesis that ferredoxin functions as a mobile shuttle in steroidogenic electron transfer, and it is considered unlikely that a functional ternary complex is formed.     

Purification of cytochrome P450 and ferredoxin, involved in bisphenol A degradation, from Sphingomonas sp. strain AO1

In a previous study (M. Sasaki, J. Maki, K. Oshiman, Y. Matsumura, and T. Tsuchido, Biodegradation 16:449-459, 2005), the cytochrome P450 monooxygenase system was shown to be involved in bisphenol A (BPA) degradation by Sphingomonas sp. strain AO1. In the present investigation, we purified the components of this monooxygenase, cytochrome P450 (P450bisd), ferredoxin (Fd(bisd)), and ferredoxin reductase (Red(bisd)). We demonstrated that P450bisd and Fd(bisd) are homodimeric proteins with molecular masses of 102.3 and 19.1 kDa, respectively, by gel filtration chromatography analysis. Spectroscopic analysis of Fd(bisd) revealed the presence of a putidaredoxin-type [2Fe-2S] cluster. P450(bisd), in the presence of Fd(bisd), Red(bisd), and NADH, was able to convert BPA. The K(m) and kcat values for BPA degradation were 85 +/- 4.7 microM and 3.9 +/- 0.04 min(-1), respectively. NADPH, spinach ferredoxin, and spinach ferredoxin reductase resulted in weak monooxygenase activity. These results indicated that the electron transport system of P450bisd might exhibit strict specificity. Two BPA degradation products of the P450(bisd) system were detected by high-performance liquid chromatography analysis and were thought to be 1,2-bis(4-hydroxyphenyl)-2-propanol and 2,2-bis(4-hydroxyphenyl)-1-propanol based on mass spectrometry-mass spectrometry analysis. This is the first report demonstrating that the cytochrome P450 monooxygenase system in bacteria is involved in BPA degradation.     

Interactions of mammalian cytochrome P450, NADPH-cytochrome P450 reductase, and cytochrome b(5) enzymes

An immobilized system was developed to detect interactions of human cytochromes P450 (P450) with the accessory proteins NADPH-P450 reductase and cytochrome b(5) (b(5)) using an enzyme-linked affinity approach. Purified enzymes were first bound to wells of a polystyrene plate, and biotinylated partner enzymes were added and bound. A streptavidin-peroxidase complex was added, and protein-protein binding was monitored by measuring peroxidase activity of the bound biotinylated proteins. In a model study, we examined protein-protein interactions of Pseudomonas putida putidaredoxin (Pdx) and putidaredoxin reductase (PdR). A linear relationship (r(2)=0.96) was observed for binding of PdR-biotin to immobilized Pdx compared with binding of Pdx-biotin to immobilized PdR (the estimated K(d) value for the Pdx.PdR complex was 0.054muM). Human P450 2A6 interacted strongly with NADPH-P450 reductase; the K(d) values (with the reductase) ranged between 0.005 and 0.1muM for P450s 2C19, 2D6, and 3A4. Relatively weak interaction was found between holo-b(5) or apo-b(5) (devoid of heme) with NADPH-P450 reductase. Among the rat, rabbit, and human P450 1A2 enzymes, the rat enzyme showed the tightest interaction with b(5), although no increases in 7-ethoxyresorufin O-deethylation activities were observed with any of the P450 1A2 enzymes. Human P450s 2A6, 2D6, 2E1, and 3A4 interacted well with b(5), with P450 3A4 yielding the lowest K(d) values followed by P450s 2A6 and 2D6. No appreciable increases in interaction between human P450s with b(5) or NADPH-P450 reductase were observed when typical substrates for the P450s were included. We also found that NADPH-P450 reductase did not cause changes in the P450.substrate K(d) values estimated from substrate-induced UV-visible spectral changes with rabbit P450 1A2 or human P450 2A6, 2D6, or 3A4. Collectively, the results show direct and tight interactions between P450 enzymes and the accessory proteins NADPH-P450 reductase and b(5), with different affinities, and that ligand binding to mammalian P450s did not lead to increased interaction between P450s and the reductase.     

Cytochrome b5 increases the rate of product formation by cytochrome P450 2B4 and competes with cytochrome P450 reductase for a binding site on cytochrome P450 2B4

The kinetics of product formation by cytochrome P450 2B4 were compared in the presence of cytochrome b(5) (cyt b(5)) and NADPH-cyt P450 reductase (CPR) under conditions in which cytochrome P450 (cyt P450) underwent a single catalytic cycle with two substrates, benzphetamine and cyclohexane. At a cyt P450:cyt b(5) molar ratio of 1:1 under single turnover conditions, cyt P450 2B4 catalyzes the oxidation of the substrates, benzphetamine and cyclohexane, with rate constants of 18 +/- 2 and 29 +/- 4.5 s(-1), respectively. Approximately 500 pmol of norbenzphetamine and 58 pmol of cyclohexanol were formed per nmol of cyt P450. In marked contrast, at a cyt P450:CPR molar ratio of 1:1, cyt P450 2B4 catalyzes the oxidation of benzphetamine congruent with100-fold (k = 0.15 +/- 0.05 s(-1)) and cyclohexane congruent with10-fold (k = 2.5 +/- 0.35 s(-1)) more slowly. Four hundred picomoles of norbenzphetamine and 21 pmol of cyclohexanol were formed per nmol of cyt P450. In the presence of equimolar concentrations of cyt P450, cyt b(5), and CPR, product formation is biphasic and occurs with fast and slow rate constants characteristic of catalysis by cyt b(5) and CPR. Increasing the concentration of cyt b(5) enhanced the amount of product formed by cyt b(5) while decreasing the amount of product generated by CPR. Under steady-state conditions at all cyt b(5):cyt P450 molar ratios examined, cyt b(5) inhibits the rate of NADPH consumption. Nevertheless, at low cyt b(5):cyt P450 molar ratios 相似文献   

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.     

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.     

Purification and characterization of 2-ethoxyphenol-induced cytochrome P450 from Corynebacterium sp. strain EP1

A soluble cytochrome P450 (P450EP1A) induced by 2-ethoxyphenol was purified to apparent homogeneity from Corynebacterium sp. strain EP1. The P450EP1A showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a molecular weight of about 45 kDa. The CO-reduced difference spectra of P450EP1A had a Soret maximum at 447.6 nm. The substrate difference spectra with 2-ethoxyphenol showed an absorption maximum at 394.0 nm. The purified P450EP1A degraded 2-ethoxyphenol in an assay system composed of spinach ferredoxin-NADP+ oxidoreductase and NADPH. The reaction activity decreased to 1.4% of its original activity by addition of CO. The existence of catechol in the reaction mixture was confirmed after the metabolic reaction, indicating that P450EP1A catalyzes O-dealkylation of 2-ethoxyphenol. In addition to 2-ethoxyphenol, the P450EP1A metabolized 2-methoxyphenol, 1,1,1-trichloroethane, carbon tetrachloride, benzene, and toluene.     

Functional expression system for cytochrome P450 genes using the reductase domain of self-sufficient P450RhF from Rhodococcus sp. NCIMB 9784

Cytochrome P450RhF from Rhodococcus sp. NCIMB 9784 is a self-sufficient P450 monooxygenase. We report here a simple system for the functional expression of various P450 genes using the reductase domain of this P450RhF, which comprises flavin mononucleotide- and nicotinamide adenine dinucleotide phosphate binding motifs and a [2Fe2S] ferredoxin-like center. Vector pRED was constructed, which carried the T7 promoter, cloning sites for a P450, a linker sequence, and the P450RhF reductase domain, in this order. The known P450 genes, encoding P450cam from Pseudomonas putida (CYP101A) and P450bzo from an environmental metagenome library (CYP203A), were expressed on vector pRED as soluble fusion enzymes with their natural spectral features in Escherichia coli. These E. coli cells expressing the P450cam and P450bzo genes could convert (+)-camphor and 4-hydroxybenzoate into 5-exo-hydroxycamphor and protocatechuate (3,4-dihydroxybenzoate), respectively (the expected products). Using this system, we also succeeded in directly identifying the function of P450 CYP153A as alkane 1-monooxygenase for the first time, i.e., E. coli cells expressing a P450 CYP153A gene named P450balk, which was isolated form Alcanivorax borkumensis SK2, converted octane into 1-octanol with high efficiency (800 mg/l). The system presented here may be applicable to the functional identification of a wide variety of bacterial cytochromes P450.     

Examining the mechanism of stimulation of cytochrome P450 by cytochrome b5: the effect of cytochrome b5 on the interaction between cytochrome P450 2B4 and P450 reductase

Dissociation constants K(d) for cytochrome P450 reductase (reductase) and cytochrome P450 2B4 are measured in the presence of various substrates. Aminopyrine increases the dissociation constant for binding of the two proteins. Furthermore, cytochrome b(5) (b(5)) stimulates metabolism of this substrate and dramatically decreases the substrate-related K(d) values. Experiments are performed to test if the b(5)-mediated stimulation is effected through a conformational change of P450. The effects of a redox-inactive analogue of b(5) (Mn b(5)) on product formation and reaction stoichiometry are determined. Variations in the concentration of Mn b(5) stock solution that have been shown to effect the aggregation state of the protein alter the rate of P450-mediated NADPH oxidation but have no effect on the rate of product formation. Thus, the electron transfer capability of b(5) is necessary for stimulation of metabolism. Furthermore, stopped flow spectrometry measurements of the rate of first electron reduction of the P450 by reductase indicate that the coupling of P450 2B4-mediated metabolism improves, in the presence of Mn b(5), with slower delivery of the first electron of the catalytic cycle by the reductase. These results are consistent with a model involving the regulation of the P450 catalytic cycle by conformational changes of the P450 enzyme. We propose that the conformational change(s) necessary for progression of the catalytic cycle is inhibited when reduced, but not oxidized, reductase is bound to the P450.     

31P NMR spectroscopic studies on purified, native and cloned, expressed forms of NADPH-cytochrome P450 reductase.

31P NMR spectroscopy has been utilized in conjunction with site-directed mutagenesis and phospholipid analysis to determine structural aspects of the prosthetic flavins, FAD and FMN, of NADPH-cytochrome P450 reductase. Comparisons are made among detergent-solubilized and protease (steapsin)-solubilized preparations of porcine liver reductases, showing unequivocally that the 31P NMR signals at approximately 0.0 ppm in the detergent-solubilized, hydrophobic form are attributable to phospholipids. By extraction and TLC analysis, the phospholipid contents of detergent-solubilized rat liver reductase, both tissue-purified and Escherichia coli-expressed, have been determined to reflect the membranes from which the enzyme was extracted. In addition, the cloned, wild-type NADPH-cytochrome P450 reductase exhibits an additional pair of signals downfield of the normal FAD pyrophosphate resonances reported by Otvos et al. [(1986) Biochemistry 25, 7220-7228], but these signals are not observed with tissue-purified or mutant enzyme preparations. The Tyr140----Asp140 mutant, which exhibits only 20% of wild-type activity, displays no gross changes in 31P NMR spectra. However, the Tyr178----Asp178 mutant, which has no catalytic activity and does not bind FMN, exhibits no FMN 31P NMR signal and a normal, but low intensity, pair of signals for FAD. The latter experiments, taking advantage of mutations in residues putatively on either side of the FMN isoalloxazine ring, suggest subtle to severe changes in the binding of the flavin prosthetic groups and, perhaps, cooperative interactions of flavin binding to NADPH-cytochrome P450 reductase.     

P450 reductase and cytochrome b5 interactions with cytochrome P450: effects on house fly CYP6A1 catalysis

The interactions of protein components of the xenobiotic-metabolizing cytochrome P450 system, CYP6A1, P450 reductase, and cytochrome b5 from the house fly (Musca domestica) have been characterized. CYP6A1 activity is determined by the concentration of the CYP6A1-P450 reductase complex, regardless of which protein is present in excess. Both holo- and apo-b5 stimulated CYP6A1 heptachlor epoxidase and steroid hydroxylase activities and influenced the regioselectivity of testosterone hydroxylation. The conversion of CYP6A1 to its P420 form was decreased by the addition of apo-b5. The effects of cytochrome b5 may involve allosteric modification of the P450 enzyme that modify the conformation of the active site. The overall stoichiometry of the P450 reaction was substrate-dependent. High uncoupling of CYP6A1 was observed with generation of hydrogen peroxide, in excess over the concomitant testosterone hydroxylation or heptachlor epoxidation. Inclusion of cytochrome b5 in the reconstituted system improved efficiency of oxygen consumption and electron utilization from NADPH, or coupling of the P450 reaction. Depending on the reconstitution conditions, coupling efficiency varied from 8 to 25% for heptachlor epoxidation, and from 11 to 70% for testosterone hydroxylation. Because CYP6A1 is a P450 involved in insecticide resistance, this suggests that xenobiotic metabolism by constitutively overexpressed P450s may be linked to significant oxidative stress in the cell that may carry a fitness cost.     

The role of cytochrome P450 lysine residues in the interaction between cytochrome P450IA1 and NADPH-cytochrome P450 reductase.

Cytochrome P450IA1 (purified from hepatic microsomes of beta-naphthoflavone-treated rats) has been covalently modified with the lysine-modifying reagent acetic anhydride. Different levels of lysine residue modification in cytochrome P450IA1 can be achieved by varying the concentration of acetic anhydride. Modification of lysine residues in P450IA1 greatly inhibits the interaction of P450IA1 with NADPH-cytochrome P450 reductase. Modification of 1.0 and 3.3 mol lysine residues per mole P450IA1 resulted in 30 and 95% decreases, respectively, in 7-ethoxycoumarin hydroxylation by a reconstituted P450IA1/reductase complex. However, modification of 3.3 mol lysine residues per mole P450IA1 decreased only cumene hydroperoxide-supported P450-dependent 7-ethoxycoumarin hydroxylation by 30%. Spectral and fluorescence studies showed no indication of global conformational change of P450IA1 even with up to 8.8 mol lysine residues modified per mole P450IA1. These data suggest that at least three lysine residues in P450IA1 may be involved in the interaction with reductase. Identification of lysine residues in P450IA1 possibly involved in this interaction was carried out by [14C]acetic anhydride modification, trypsin digestion, HPLC separation, and amino acid sequencing. The lysine residue candidates identified in this manner were K97, K271, K279, and K407.     

Interflavin electron transfer in human cytochrome P450 reductase is enhanced by coenzyme binding. Relaxation kinetic studies with coenzyme analogues.

The role of coenzyme binding in regulating interflavin electron transfer in human cytochrome P450 reductase (CPR) has been studied using temperature-jump spectroscopy. Previous studies [Gutierrez, A., Paine, M., Wolf, C.R., Scrutton, N.S., & Roberts, G.C.K. Biochemistry (2002) 41, 4626-4637] have shown that the observed rate, 1/tau, of interflavin electron transfer (FADsq - FMNsq-->FADox - FMNhq) in CPR reduced at the two-electron level with NADPH is 55 +/- 2 s-1, whereas with dithionite-reduced enzyme the observed rate is 11 +/- 0.5 s-1, suggesting that NADPH (or NADP+) binding has an important role in controlling the rate of internal electron transfer. In relaxation experiments performed with CPR reduced at the two-electron level with NADH, the observed rate of internal electron transfer (1/tau = 18 +/- 0.7 s-1) is intermediate in value between those seen with dithionite-reduced and NADPH-reduced enzyme, indicating that the presence of the 2'-phosphate is important for enhancing internal electron transfer. To investigate this further, temperature jump experiments were performed with dithionite-reduced enzyme in the presence of 2',5'-ADP and 2'-AMP. These two ligands increase the observed rate of interflavin electron transfer in two-electron reduced CPR from 1/tau = 11 s-1 to 35 +/- 0.2 s-1 and 32 +/- 0.6 s-1, respectively. Reduction of CPR at the two-electron level by NADPH, NADH or dithionite generates the same spectral species, consistent with an electron distribution that is equivalent regardless of reductant at the initiation of the temperature jump. Spectroelectrochemical experiments establish that the redox potentials of the flavins of CPR are unchanged on binding 2',5'-ADP, supporting the view that enhanced rates of interdomain electron transfer have their origin in a conformational change produced by binding NADPH or its fragments. Addition of 2',5'-ADP either to the isolated FAD-domain or to full-length CPR (in their oxidized and reduced forms) leads to perturbation of the optical spectra of both the flavins, consistent with a conformational change that alters the environment of these redox cofactors. The binding of 2',5'-ADP eliminates the unusual dependence of the observed flavin reduction rate on NADPH concentration (i.e. enhanced at low coenzyme concentration) observed in stopped-flow studies. The data are discussed in the context of previous kinetic studies and of the crystallographic structure of rat CPR.     

Optimization of yeast-expressed human liver cytochrome P450 3A4 catalytic activities by coexpressing NADPH-cytochrome P450 reductase and cytochrome b5.

Human liver P450 NF25 (CYP3A4) had been previously expressed in Saccharomyces cerevisiae using the inducible GAL10-CYC1 promoter and the phosphoglycerate kinase gene terminator [Renaud, J. P., Cullin, C., Pompon, D., Beaune, P. and Mansuy, D. (1990) Eur. J. Biochem. 194, 889-896]. The use of an improved expression vector [Urban, P., Cullin, C. and Pompon, D. (1990) Biochimie 72, 463-472] increased the amounts of P450 NF25 produced/culture medium by a factor of five, yielding up to 10 nmol/l. The availability of recently developed host cells that simultaneously overexpress yeast NADPH-P450 reductase and/or express human liver cytochrome b5, obtained through stable integration of the corresponding coding sequences into the yeast genome, led to biotechnological systems with much higher activities of yeast-expressed P450 NF25 and with much better ability to form P450 NF25-iron-metabolite complexes. 9-fold, 8-fold, and 30-fold rate increases were found respectively for nifedipine 1,4-oxidation, lidocaine N-deethylation and testosterone 6 beta-hydroxylation between P450 NF25-containing yeast microsomes from the basic strain and from the strain that both overexpresses yeast NADPH-P450 reductase and expresses human cytochrome b5. Even higher turnovers (15-fold, 20-fold and 50-fold rate increases) were obtained using P450 NF25-containing microsomes from the yeast just overexpressing yeast NADPH-P450 reductase in the presence of externally added, purified rabbit liver cytochrome b5. This is explained by the fact that the latter strain contained the highest level of NADPH-P450 reductase activity. It is noteworthy that for the three tested substrates, the presence of human or rabbit cytochrome b5 always showed a stimulating effect on the catalytic activities and this effect was saturable. Indeed, addition of rabbit cytochrome b5 to microsomes from a strain expressing human cytochrome b5 did not further enhance the catalytic rates. The yeast expression system was also used to study the formation of a P450-NF25-iron-metabolite complex. A P450 Fe(II)-(RNO) complex was obtained upon oxidation of N-hydroxyamphetamine, catalyzed by P450-NF25-containing yeast microsomes. In microsomes from the basic strain expressing P450 NF25, 10% of the starting P450 NF25 was transformed into this metabolite complex, whereas more than 80% of the starting P450 NF25 led to complex formation in microsomes from the strain overexpressing yeast NADPH-P450 reductase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Highly purified cytochrome P-448 and P=450 from rat liver microsomes.

Cytochrome P-448 from 3-methylcholanthrene-treated rats has been purified to a specific content of greater than 20 nmoles/mg protein, and cytochrome P-450 from phenobarbital-treated rats to greater than 17 nmoles/mg protein. Both cytochromes are catalytically active when reconstituted with lipid and NADPH-cytochrome c reductase and exhibit differential substrate specificities for benzphetamine and benzo[a]pyrene. Cytochrome P-448 has a minimum molecular weight of approximately 53,000, and cytochrome P-450, 48,000 by SDS polyacrylamide gel electrophoresis.     

Characterization of three cytochrome P450s purified from renal microsomes of untreated male rats and comparison with human renal cytochrome P450

Three renal cytochrome P450s (P450 K-2, K-4, and K-5) were purified from renal microsomes of untreated male rats. Also, the human renal cytochrome P450 (P450 HK) was partially purified from renal microsomes and its properties were compared with those of the rat renal cytochrome P450s. The molecular weight of P450 K-2, K-4, and K-5 was 52,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The absolute spectrum of the oxidized forms indicated that they had the low-spin state of heme, and the CO-reduced spectral maxima of P450 K-2, K-4, and K-5 were at 449, 451, and 452 nm, respectively. NH2-terminal sequence analysis of P450 K-2, K-4, and K-5 showed that these forms were different from hepatic cytochrome P450s purified previously. P450 K-2, K-4, and K-5 catalyzed the O-dealkylation of 7-ethoxycoumarin but were not efficient in the hydroxylation of testosterone. Aminopyrine was metabolized by P450 K-2 and K-4 but not by P450 K-5. Lauric acid was metabolized efficiently by all of these forms in the presence of cytochrome b5. The regiospecificity of these forms toward lauric acid was different. P450 K-2 hydroxylated lauric acid only at the (omega-1)-position, not at the omega-position. P450 K-4 and K-5 hydroxylated lauric acid at both the omega- and (omega-1)-positions. The ratios of omega/(omega-1)-hydroxylation activity of P450 K-4 and K-5 were 2.5 and 7.8, respectively. Human P450 HK was purified 220-fold and its specific content was 2.0 nmol/mg of protein. The Soret maxima of P450 HK were at 418 nm for the oxidized form, 416 nm for the reduced form, and 450 nm for the CO-reduced form. P450 HK catalyzed the O-dealkylation of 7-ethoxycoumarin but was not efficient in aminopyrine N-demethylation or testosterone hydroxylation. P450 HK had high lauric acid omega- and (omega-1)-hydroxylation activities in the presence of cytochrome b5, especially omega-hydroxylation. These properties resembled those of P450 K-5 most closely. Anti-P450 K-5 antibody cross-reacted with P450 HK as well as P450 K-5 and only one band was stained on immunostained Western blotting for partially purified P450 HK. The molecular weight of P450 HK was 52,000 on Western blotting.     

Isolation of cytochrome P450 from hepatopancreas microsomes of the spiny lobster, Panulirus argus, and determination of catalytic activity with NADPH cytochrome P450 reductase from vertebrate liver

One major form of cytochrome P450 has been isolated from the hepatopancreas of untreated spiny lobsters, Panulirus argus. This form, termed here D1, was purified to a specific content of 12.1 +/- 1.8 nmol/mg protein. Two minor forms, termed D2 and D3 were partially purified to 4.6 +/- 1.6 and 2.3 +/- 0.2 nmol P450/mg protein, respectively. No NADPH-cytochrome P450 reductase activity was detected in spiny lobster hepatopancreas microsomes and no purification of spiny lobster reductase was achieved in this study. Very low NADPH-cytochrome c reductase activity was found in hepatopancreas microsomes and also in cytosol. Indirect evidence suggested that proteolysis of spiny lobster P450 reductase during the preparation of hepatopancreas microsomes may in part account for the lack of detectable monooxygenase activity in hepatopancreas microsomes. The catalytic activities of the D1 or D2 forms of spiny lobster P450 were measured by mixing D1 or D2 with NADPH-cytochrome P450 reductase isolated from pig or rat liver microsomes. D2 was very efficient in demethylating benzphetamine, with a turnover number of 122 per minute, and D1 was an efficient catalyst of progesterone 16 alpha-hydroxylation, with a turnover number of 43 per minute. Other good substrates for D1 and D2 forms were aminopyrine, testosterone, benzo(a)pyrene, and 7-ethoxycoumarin. Little activity was found with methyl-, ethyl-, pentyl-, or benzyl-phenoxazone ethers as substrates. The profile of metabolites formed by D1 or D2 with benzo(a)pyrene as substrate were more similar to those formed with uninduced rat liver microsomes than to those formed by liver microsomes from uninduced flatfish species.     

Kinetic and spectroscopic probes of motions and catalysis in the cytochrome P450 reductase family of enzymes

There is a mounting body of evidence to suggest that enzyme motions are linked to function, although the design of informative experiments aiming to evaluate how this motion facilitates reaction chemistry is challenging. For the family of diflavin reductase enzymes, typified by cytochrome P450 reductase, accumulating evidence suggests that electron transfer is somehow coupled to large-scale conformational change and that protein motions gate the electron transfer chemistry. These ideas have emerged from a variety of experimental approaches, including structural biology methods (i.e. X-ray crystallography, electron paramagnetic/NMR spectroscopies and solution X-ray scattering) and advanced spectroscopic techniques that have employed the use of variable pressure kinetic methodologies, together with solvent perturbation studies (i.e. ionic strength, deuteration and viscosity). Here, we offer a personal perspective on the importance of motions to electron transfer in the cytochrome P450 reductase family of enzymes, drawing on the detailed insight that can be obtained by combining these multiple structural and biophysical approaches.