The stoichiometry of the cytochrome P-450-catalyzed metabolism of methoxyflurane and benzphetamine in the presence and absence of cytochrome b5.

The complete stoichiometry of the metabolism of the cytochrome b5 (cyt b5)-requiring substrate, methoxyflurane, by purified cytochrome P-450 2B4 was compared to that of another substrate, benzphetamine, which does not require cyt b5 for its metabolism. Cyt b5 invariably improved the efficiency of product formation. That is, in the presence of cyt b5 a greater percentage of the reducing equivalents from NADPH were utilized to generate substrate metabolites, primarily at the expense of the side product, superoxide. With methoxyflurane, cyt b5 addition always resulted in an increased rate of product formation, while with benzphetamine the rate of product formation remained unchanged, increased or decreased. The apparently contradictory observations of increased reaction efficiency but decrease in total product formation for benzphetamine can be explained by a second effect of cyt b5. Under some experimental conditions cyt b5 inhibits total NADPH consumption. Whether stimulation, inhibition, or no change in product formation is observed in the presence of cyt b5 depends on the net effect of the stimulatory and inhibitory effects of cyt b5. When total NADPH consumption is inhibited by cyt b5, the rapidly metabolized, highly coupled (approximately equal to 50%) substrate, benzphetamine, undergoes a net decrease in metabolism not counterbalanced by the increase in the efficiency (2-20%) of the reaction. In contrast, in the presence of the slowly metabolized, poorly coupled (approximately equal to 0.5-3%) substrate, methoxyflurane, inhibition of total NADPH consumption by cyt b5 was never sufficient to overcome the stimulation of product formation due to an increase in efficiency of the reaction.

The complete stoichiometry of the metabolism of the cytochrome b 5 (cyt b 5 )-requiring substrate, methoxyflurane, by purified cytochrome P-450 2B4 was compared to that of another substrate, benzphetamine, which does not require cyt b 5 for its metabolism. Cyt b 5 invariably improved the efficiency of product formation. That is, in the presence of cyt b 5 a greater percentage of the reducing equivalents from NADPH were utilized to generate substrate metabolites, primarily at the expense of the side product, superoxide.
With methoxyflurane, cyt b 5 addition always resulted in an increased rate of product formation, while with benzphetamine the rate of product formation remained unchanged, increased or decreased. The apparently contradictory observations of increased reaction efficiency but decrease in total product formation for benzphetamine can be explained by a second effect of cyt b 5 . Under some experimental conditions cyt b 5 inhibits total NADPH consumption. Whether stimulation, inhibition, or no change in product formation is observed in the presence of cyt b 5 depends on the net effect of the stimulatory and inhibitory effects of cyt b 5 . When total NADPH consumption is inhibited by cyt b 5 , the rapidly metabolized, highly coupled (Х50%) substrate, benzphetamine, undergoes a net decrease in metabolism not counterbalanced by the increase in the efficiency (2-20%) of the reaction. In contrast, in the presence of the slowly metabolized, poorly coupled (Х0.5-3%) substrate, methoxyflurane, inhibition of total NADPH consumption by cyt b 5 was never sufficient to overcome the stimulation of product formation due to an increase in efficiency of the reaction.
It has previously been demonstrated that the O-demethylation of the volatile anesthetic methoxyflurane (CHCl 2 CF 2 OCH 3 ) by the major phenobarbital-inducible hepatic cytochrome P-450 2B4 of rabbit is markedly stimulated in the presence of cytochrome b 5 (Canova-Davis et al., 1985;Canova-Davis and Waskell, 1984). Cytochrome b 5 is believed to function in this reaction by providing the second of the two electrons required for substrate oxidation by cytochrome P-450. The first electron is donated by cytochrome P-450 reductase.
An intriguing long standing question has been why is the metabolism of methoxyflurane and a minority of other substrates, such as nifedipine, p-nitroanisole, prostaglandin, lauric acid, n-methylcarbazole, chlorobenzene, p-nitrophenetole, 7-ethoxycoumarin, benzo(a)pyrene, and lidocaine, and testosterone ␤-hydroxylation so stimulated by the presence of cytochrome b 5 while the metabolism of most other substrates, including benzphetamine, is minimally stimulated, not effected, or slightly inhibited in the presence of cytochrome b 5 (Aoyama et al., 1990;Canova-Davis and Waskell, 1984;Hoffman et al., 1989;Okita et al., 1981;Peyronneau et al., 1992;Vatsis et al., 1982).
The answer to this question depends on understanding the individual steps of the catalytic reaction cycle of cytochrome P-450: 1) binding of substrate; 2) one electron reduction of the ferric substrate-enzyme complex by cytochrome P-450 reductase; 3) the binding of oxygen to the ferrous enzyme. In uncoupled reactions oxygen can dissociate from the oxyferrous enzyme to regenerate the ferric cytochrome and superoxide anion which in turn can dismutate to hydrogen peroxide according to Equation 1: 4) transfer of a second electron from either cytochrome P-450 reductase or cytochrome b 5 ; 5) protonation of the distal oxygen atom by a single hydrogen ion. In uncoupled reactions two protons can be provided at this step to directly generate hydrogen peroxide and the ferric enzyme. This is the oxidase activity of cytochrome P-450 and consumes 1 equivalent of oxygen and NADPH; O 2 ϩ NADPH ϩ H ϩ 3 H 2 O 2 ϩ NADP ϩ (Eq. 2) 6) cleavage of the oxygen-oxygen bond with formation of a high valency iron-oxo intermediate [FeϭO] 3ϩ and water. In uncoupled reaction cycles it is presumed that this iron-oxo intermediate can undergo further two-electron reduction by consuming a second molecule of NADPH to generate a second, "extra" molecule of water to distinguish it from the water which is produced in an equal amount to product in the monooxidation reaction (Equation 3); 7) insertion of the second oxygen atom now an "activated oxygen" into a carbon-hydrogen bond of the substrate; and 8) dissociation of the product to regenerate the ferric enzyme (White, 1991;White and Coon, 1980). The stoichiometry of eukaryotic microsomal cytochrome P-450-catalyzed reactions has been studied extensively both in microsomes and in the reconstituted system (Gorsky and Coon, 1986;Gorsky et al., 1984;Nordblom and Coon, 1977;Zhukov and Archakov, 1982). If the cytochrome P-450-catalyzed reactions were perfectly coupled, they would occur as shown in Equation 3, where RH represents substrate and ROH the oxidized product.
However, the eukaryotic microsomal cytochrome P-450 reaction cycle usually becomes uncoupled at one or more of the three theoretically possible steps to produce the one-, two-, and four-electron-reduced species of oxygen, i.e. superoxide (Ingelman-Sundberg and Johansson, 1980;Kuthan et al., 1978), hydrogen peroxide (Heinemeyer et al., 1980;Oprian et al., 1983), and water (Gorsky et al., 1984;Morgan et al., 1982;Zhukov and Archakov, 1982), respectively. In in vitro reactions, hydrogen peroxide arises in two ways: by dismutation of the superoxide anion (Equation 1) and by dissociation of hydrogen peroxide from a two-electron-reduced cytochrome P-450.
The stoichiometry of the oxidation of camphor by cytochrome P-450 camphor is completely coupled. However, the reaction is highly uncoupled when either the wild type enzyme oxidizes substrates other than camphor or selected mutant cytochromes P-450 oxidize camphor Sligar, 1992, 1994;Imai et al., 1989;Loida and Sligar, 1993;Martinis et al., 1989;Poulos and Howard, 1987;Raag et al., 1993;Raag and Poulos, 1989). In contrast to the eukaryotic cytochromes P-450, autooxidation of wild type cytochrome P-450 camphor to superoxide is much slower (0.004 s Ϫ1 at 11°C) than the introduction of the second electron (Х5 s Ϫ1 ) and steady-state cycling of the system. However, in contrast to the microsomal enzymes, hydrogen peroxide is produced in uncoupled cytochrome P-450 camphor reactions by the autooxidation of the two-electron-reduced oxycytochrome P-450 camphor complex. Either water in the active site or a protonated active site amino acid is assumed to provide the necessary hydrogen ions which allow the two-electronreduced oxygen to dissociate as hydrogen peroxide and regenerate the ferric protein (Gerber and Sligar, 1994;Swinney and Mak, 1994). Since hydrogen peroxide is produced only in the absence of strict complementarity between the substrate and the proteinaceous substrate binding site, steric factors near the two-electron-reduced heme-oxygen complex are presumably altered in such a way to facilitate protonation and dissociation of the reduced oxygen rather than protonation with cleavage of the oxygen bond to yield water and the reactive oxidizing species [FeϭO] 3ϩ (Atkins and Sligar, 1987;Martinis et al., 1989). In addition to providing protons, water in the substratebinding site would increase the polarity of the pocket and favor separation of the hydrogen peroxide from the heme (Loida and Sligar, 1993). Whether the iron-oxo species oxidizes the substrate or undergoes further reduction to water depends on the proximity and orientation of the substrate to the iron-oxo species, the heat of formation of the radical formed by abstracting a hydrogen, and proton availability.
The stoichiometry experiments described in this article were undertaken with substrates and a pseudosubstrate, enflurane, in an effort to delineate the precise effect of cytochrome b 5 in cytochrome P-450-catalyzed reactions. Previous work has indicated that the activity and relative stoichiometry observed in a reconstituted system is influenced by such variables as the protein-to-lipid ratio (Bösterling et al., 1982), the length of preincubation (Causey et al., 1990), and the order of addition of the reactants (Gorsky and Coon, 1986). For this reason the reactions were conducted under a variety of conditions to assure ourselves of the generality of observed differences. In this study we report that cytochrome b 5 consistently decreases superoxide production during the metabolism of the two substrates methoxyflurane and benzphetamine, and under "Discussion" we speculate about the possible molecular basis for this effect.
[ 2 H 4 ]Methanol (99% 2 H) was obtained from Stohler Isotope Chemicals. Other chemicals used were reagent grade. Solutions of DLPC in water (1.0 mg/ml) were sonicated in a bath sonicator until clear (about 15 min).

Purification of Microsomal Enzymes
Our studies with rabbits were approved by our institution's Animal Welfare Committee. Liver microsomes were prepared from phenobarbital-treated white New Zealand male rabbits as described by Haugen and Coon (1976). Cytochrome P-450 2B4 was purified from rabbit liver microsomes as described previously (Canova-Davis and Waskell, 1984). The concentration of cytochrome P-450 was determined by the method of Omura and Sato (1964) using an extinction coefficient of 91 mM Ϫ1 cm Ϫ1 . The specific content of the purified cytochrome P-450 was 12-13 nmol/mg of protein. Cytochrome b 5 was purified from detergent-solubilized rabbit liver microsomes using the method of Chiang (1981) and had a specific content of 29 -50 nmol/mg of protein. The concentration of the purified cytochrome b 5 was determined from the absolute spectrum of the Fe(III) protein using an absorption coefficient of 117 mM Ϫ1 cm Ϫ1 at 413 nm (Strittmatter and Velick, 1956). NADPH-cytochrome P-450 reductase was purified according to the method of Yasukochi and Masters (1976) from rabbit liver microsomes prepared by the calcium precipitation method of Cinti et al. (1972). The preparations used varied widely in their specific activity, catalyzing the reduction of from 15 to 57 mol of cytochrome c/min/mg of protein in 0.3 M potassium phosphate buffer at 30°C (French and Coon, 1979). The concentration of the reductase preparations was calculated from their activity in the cytochrome c assay with the assumption that pure reductase reduces 55 mol of cytochrome c/min/mg of the protein under the above assay conditions.

Analytical Methods
Determination of the Superoxide Radical Anion-Superoxide was measured spectrophotometrically by observing its ability to reduce succinoylated ferric cytochrome c prepared by the method described by Kuthan et al. (1982). The procedure yields a mixture of partially succinoylated cytochrome c molecules with residual ability to be completely reduced by the reductase in a biphasic manner with rate constants typically of 0.65 min Ϫ1 and 0.18 min Ϫ1 . Native cytochrome c on the other hand is reduced by reductase 1000-fold faster. None of our succinoylated cytochrome c preparations contained any cytochrome c that could be reduced at the same rate as unmodified cytochrome c. It was therefore concluded that 100% of the cytochrome c had been at least partially succinoylated and its reaction with the reductase markedly inhibited. The reduction of cytochrome c was measured at 550 nm using an extinction coefficient of 21 mM Ϫ1 cm Ϫ1 . Besides reacting with succinoylated cytochrome c the superoxide produced in the reconstituted system may also spontaneously dismutate or undergo side reactions with the components of the reconstituted system. Therefore, the experimentally determined rate of reduction of succinoylated cytochrome c will always be less than the actual amount of superoxide produced. In order to determine the actual amount of superoxide produced, Equation 23 was derived as described under "Appendix." This equation relates the experimentally determined rate of superoxide production to the actual rate of superoxide production while accounting for the side reactions of superoxide. Equation 23, however, has three unknowns: 1) V max , the actual rate of superoxide production; 2) F, the ratio of a constant for the reaction between superoxide and the reaction components to the rate constant for the reaction of superoxide with succinoylated cytochrome c (see Equations 5, 11, and 18); and 3) K, the ratio of a constant which reflects the rate of dismutation to the square of the rate constant for the reaction of superoxide with succinoylated cytochrome c (Equations 5,8,and 17).
The three unknowns, V max , K, and F, were obtained following acquisition of experimental data which was curve fit as follows. The rate of reduction of succinoylated cytochrome c produced under a particular set of conditions in the reconstituted system was measured at four different cytochrome c concentrations. This procedure was performed for each set of experimental conditions used. The concentration of cytochrome c, typically 14 -72 M, was high enough to keep the cytochrome c concentration constant throughout the reaction, but not so high as to undergo significant reactions with cytochrome b 5 and cytochrome P-450 reductase. The reaction was usually measured for 30 s at a cytochrome P-450 concentration of 0.2 M when superoxide was produced at a rate of approximately 40 nmol/nmol of cytochrome P-450/min or less. Insufficient hydrogen peroxide is formed under these conditions to reoxidize succinoylated cytochrome c. Depending on the reaction conditions, 4.5-70% of the rate of reduction of succinoylated cytochrome c could not be inhibited by addition of superoxide dismutase to the reconstituted system and was therefore assumed to be the result of the direct reduction by cytochrome b 5 and cytochrome P-450 reductase. The experimentally determined rate of succinoylated cytochrome c reduction as a function of four different cytochrome c concentrations served as the experimental data which were fit to Equation 23 (using the Marquardt-Levenberg algorithm of Sigma Plot), allowing the parameters K, V max , and F to vary until the best fit of data and theoretical curve was found. At this point the three unknown parameters were assumed to be the actual values, i.e. V max was considered to be the true rate of superoxide generation in the reconstituted system. The V max calculated with each batch of succinoylated cytochrome c with superoxide generated in the xanthine/xanthine oxidase system (McCord and Fridovich, 1968) was slightly less (10 -30%) than the experimentally determined V max using unmodified cytochrome c. Therefore, in order to obtain the true amount of superoxide produced, the results obtained with each batch of succinoylated cytochrome c were normalized to the results obtained with native cytochrome c. Side reactions of superoxide with the reaction components were minimized by using only those reagents and protein preparations which did not react with superoxide, i.e. depress the reduction of succinoylated cytochrome c by superoxide generated in the xanthine/xanthine oxidase system. Addition of 1 M Fe 3ϩ to the reaction mixture did not affect the amount of superoxide measured whereas concentrations of Fe 3ϩ greater than 1 M decreased the amount of superoxide measured.
Measurement of Methoxydifluoroacetic and Dichloroacetic Acids-The products of methoxyflurane metabolism, methoxydifluoroacetic and dichloroacetic acids, were measured by a gas chromatographicmass spectrometric assay developed in this laboratory for these experiments.
Sodium methoxydifluoroacetate was synthesized using a procedure adapted from a published report (Selinsky et al., 1988a). Sodium (2.2 g, 96 nmol) was dissolved in 20.0 ml of methanol to generate sodium methoxide. Next, 3.82 ml (5.2 g, 40 nmol) of chlorodifluoroacetic acid were added. The mixture was heated at 75°C for 30 h, and then the solvent was evaporated to give a semisolid mass of the crude product, sodium methoxydifluoroacetate. Excess 6 N sulfuric acid was added to generate the free acid, and the resulting mixture was extracted twice with 20-ml portions of ether. The ether extracts containing the methoxydifluoroacetic acid were titrated with saturated sodium bicarbonate until the evolution of CO 2 ceased, in order to regenerate the sodium salt. The aqueous phase was removed and subsequently evaporated. The remaining white solid was dissolved in hot methanol and filtered, and the methanol was evaporated to give 2.7 g (46%) of crude sodium methoxydifluoroacetate. After three recrystallizations from methanol, a pure sample giving the correct carbon and hydrogen analysis for C 3 H 3 O 2 F 2 Na was obtained. This product was characterized by NMR (Selinsky et al., 1988a) and Fourier-transformed infrared spectroscopy. The purity of the sodium methoxydifluoroacetate was confirmed by gas chromatography of an acidified ether extract. Peaks greater than 1% of the methoxydifluoroacetic acid peak were not observed by gas chromatography after methylation of the methoxydifluoroacetate in the ether extract with diazomethane. A similar procedure was used to prepare the potassium salt of [ 2 H 3 ]methoxydifluoroacetic acid using perdeuterated methanol.
The reaction mixture supernatants were analyzed for methoxydifluoroacetic and dichloroacetic acids as follows. A known amount of the internal standard, 1.0 g of [ 2 H 3 ]methoxydifluoroacetic acid and 0.4 g of 2,2-[2-13 C]dichloroacetic acid in 5 l of water, was added to a 100 -200-l aliquot of the reaction mixture. The anesthetic was removed by placing the reaction mixture aliquot in a vacuum oven at room temperature and under 10 mm Hg pressure for 15 min. Ether-soluble impurities and traces of the anesthetic were removed by extracting the aqueous solution once with 1.0 ml of ether. Sulfuric acid (100 l, 3.0 M) was added to the aqueous phase to convert the organic acid salts to the free acids which were then extracted with a second 1.0-ml portion of ether. The ether extracts were carefully evaporated just to dryness, and methyl esters were formed by adding 2 drops of ethereal diazomethane.
Selected ion monitoring gas chromatography-mass spectrometry was carried out using a Hewlett-Packard 5890 gas chromatograph coupled to a model 5971 mass spectrometer with an open split interface. A 10 m ϫ 0.53-mm GS-Q column (J&W Scientific) was used with a carrier gas (helium) flow of 5 ml/min. The column was temperature-programmed starting at 95°C with an increase of 35°C/min to 150°C. The temperature was then increased 9°C/min to 173°C and then at 2°C/min to a final temperature of 178°C. The injector temperature was held at 200°C, and the detector temperature was 280°C. Retention times were 3.5 min for methoxydifluoroacetic acid methyl ester and 5.7 min for the methyl ester of dichloroacetic acid. The methoxydifluoroacetic acid methyl ester peak did not overlap with the dichloroacetic acid methyl ester peak. The base peaks (CH 3 -O-CF 2 ϩ ) were monitored at m/z 81.1 and 84.1 for the methyl esters of methoxydifluoroacetic acid and [ 2 H 3 ]methoxydifluoroacetic acid, respectively. The fragment ion CHCl 2 ϩ at m/z 83.1 and 84.1 was monitored for dichloroacetic acid methyl ester and its internal standard.
Determination of Benzphetamine and Dimethylaniline Metabolism; Analytical Method for Formaldehyde-The N-demethylation of N,Ndimethylaniline and benzphetamine was determined by measuring the production of formaldehyde by the Nash (1953) procedure.
Assay of Hydrogen Peroxide-Hydrogen peroxide was determined by the ferrithiocyanate method (Ovenston and Parker, 1949). Standard curves were generated with known amounts of H 2 O 2 added to the appropriate control mixtures. The loss of H 2 O 2 was insignificant after incubation of reaction mixtures with H 2 O 2 under the standard experimental conditions and in the presence of up to 100 M Fe 3ϩ . When NADPH was present, the amount of hydrogen peroxide present at the end of the incubation period was equivalent to the sum of the amount added plus the amount expected to be produced under the reaction conditions.
NADPH Consumption (Spectrophotometrically and by ␣-Ketoglutarate Production)-NADPH consumption was followed in two ways, depending on the reaction being studied: 1) spectrophotometrically at 340 nm using an extinction coefficient of 6.22 mM Ϫ1 cm Ϫ1 and 2) by determination of the amount of ␣-ketoglutarate formed in the presence of an isocitrate dehydrogenase regenerating system. ␣-Ketoglutarate was analyzed spectrophotometrically as the 2,4-dinitrophenylhydrazone (Friedmann, 1957). Since the assay is slightly nonlinear, calibration standards must closely bracket the unknown ␣-ketoglutarate concentration. A 200-l aliquot of the reaction mixture was added to 20 l of 70% trichloroacetic acid, followed by 100 l of a 20 mg/100 ml (1.0 mM) solution of 2,4-dinitrophenylhydrazine in 1.0 N HCl (Sigma color reagent). After 20 min at room temperature, 500 l of 10% aqueous sodium hydroxide were added. This was allowed to stand at room temperature for an additional 10 min and was then centrifuged before determining the absorption at 440 nm.
Oxygen Consumption-Oxygen was measured with a Clark-type electrode (Yellow Springs Instrument Co.), using the Instech model 102 B with a 600-l chamber.
Measurement of Fluoride Ion Levels-Fluoride ion concentration was determined using an Orion fluoride ion-specific electrode.
Determination of Non-heme Iron-Levels of non-heme iron were measured spectrophotometrically using ferrozine (Carter, 1971). Trace amounts of ferric ion were removed from all solutions including the phosphate buffers by treatment with chelating resin. When necessary, adventitious iron was reduced in protein preparations to less than 2 M by addition of 1 mM deferoxamine and subsequent extensive dialysis to remove the chelated iron so that the final non-heme iron concentration in the reaction mixture was less than 1 M.
Assay of Protein Concentration-Protein concentration was measured according to Lowry et al. (1951) after precipitation of the proteins in the presence of trichloroacetic acid and deoxycholate (Peterson, 1977). Bovine serum albumin was used as a standard.

General Procedures for Determining Reaction Stoichiometry
For each set of reaction conditions a concentrated mixture containing cytochrome P-450, reductase, cytochrome b 5 when present, and DLPC was prepared and preincubated at room temperature for the indicated time. The order of addition of the components to this preincubation mixture was critical and varied in many of the experiments. Hence in subsequent sections the order of addition of the proteins and lipid is indicated by the order in which they are described. Typically it is 1) cytochrome P-450, 2) reductase, 3) cytochrome b 5 , and 4) DLPC. At the end of the preincubation period potassium phosphate buffer, pH 7.4, water, and substrate were added. Methoxyflurane and enflurane were added as the pure liquid (final concentration 1.0 mg/ml) and benzphetamine and dimethylaniline as 10 mM aqueous solutions (final concentration, 1.0 mM). Final concentrations of potassium phosphate buffer and of DLPC were 50 mM and 30 g/ml (47 M), respectively. If a NADPH-generating system was used, the components were added to their final concentrations of 1.2 mM DL-isocitrate, 10 mM magnesium chloride, and 0.5 unit/ml pig heart isocitrate dehydrogenase or 0.1 unit/ml E. coli enzyme. All reactions were run at 30°C. After thermal equilibration, reactions were initiated by the addition of NADPH except in experiments with pig heart isocitrate dehydrogenase. With the pig heart isocitrate dehydrogenase system, the complete regenerating system was added to the thermally equilibrated reaction mixture outside of the oxygen chamber. This reaction mixture was quickly added to the oxygen chamber, and the oxygen consumption was immediately recorded. The oxygen concentration at zero time was determined by extrapolation of the latter portion of the curve back to zero time. Samples were taken for NADPH, hydrogen peroxide, and product analysis at this time and at the end of the reaction period. NADPH was added to a final concentration of 0.3 mM in all experiments. Negative control reactions were performed in the absence of cytochrome P-450 or in the absence of NADPH.
When analysis of hydrogen peroxide, ␣-ketoglutarate, or formaldehyde was required, aliquots of the reaction mixture were added to trichloroacetic acid (final concentration, 2-7%). Generally, aliquots of 100 l were taken for hydrogen peroxide analysis, and aliquots of 200 l were taken for ␣-ketoglutarate or formaldehyde analysis. When analysis of fluoride ion or the haloacids (methoxydifluoroacetic and dichloroacetic acids) was required, the reaction was stopped by heating aliquots, to which an internal standard had been added, in a 95°C water bath for 2 min. The precipitated protein was removed by centrifugation (10,000 ϫ g for 5 min), and the supernatant was assayed for haloacid metabolites of methoxyflurane, as described previously.

Individual Reaction Conditions
Initial Rate Conditions-In these experiments, the final protein concentrations were 0.15 M P-450, 0.075 M reductase, and 0.15 M cytochrome b 5 (when present). Reactions were initiated with the addition of NADPH and after 5-min aliquots were removed for analysis of hydrogen peroxide and product formation. Oxygen consumption was measured in a separate but parallel reaction mixture. The reaction mixture was added to the 600-l oxygen chamber, and the recording of the oxygen concentration was allowed to stabilize. The reaction was started by the addition of a 6-l aliquot of NADPH directly to the chamber. Two types of initial rate experiments were performed which differed in the concentration of the cytochrome b 5 stock solution and in the method of preincubation: 1) initial rate with cytochrome b 5 from a dilute (0.16 M) solution (P-450 ϩ reductase ϩ DLPC ϩ cytochrome b 5 ). Cytochrome P-450 and reductase were mixed with DLPC and preincubated for 5 min. Potassium phosphate buffer, pH 7.4, containing substrate or the buffer containing 0.16 M cytochrome b 5 and substrate was then added. 2) Initial rate with cytochrome b 5 from a concentrated (3.4 M) solution (P-450 ϩ reductase ϩ cytochrome b 5 ϩ DLPC ϭ "normal addition sequence"). The 3.4 M solution of cytochrome b 5 was added to the mixture of cytochrome P-450 and reductase prior to the addition of phospholipid, and the preparations were preincubated for 2 h before use.
Reactions with Inverse Addition of Cytochrome b 5 -Two types of experiments were done which differed only in the sequence of addition of proteins and the time of preincubation: 1) regenerating system (pig heart) with normal addition sequence (P-450 ϩ reductase ϩ cytochrome b 5 from a concentrated solution ϩ DLPC) were added in the order indicated and the mixture was preincubated for 15 min and 2) regenerating system (pig heart) with inverse addition sequence (P-450 ϩ cytochrome b 5 from a concentrated solution ϩ reductase ϩ DLPC). Protein solutions were added in the order indicated and the mixture was preincubated for 5 min.
After the preincubation, potassium phosphate buffer, magnesium chloride, water, and the substrate were added. The reaction mixture was thermally equilibrated in a 30°C water bath for 1 min and then the NADPH-regenerating system (with NADPH) was added. The final concentration of P-450, reductase, and cytochrome b 5 (when present) was 0.2 M with methoxyflurane or without substrate and 0.1 M with benzphetamine as substrate. A portion of the reaction mixture was immediately loaded into the oxygen chamber, and after stabilization of the recording of oxygen concentration (1-2 min), the rate of oxygen consumption was determined from the linear section of the traces. The remaining portion of the reaction mixture was aliquoted for the hydrogen peroxide, ␣-ketoglutarate, and product (formaldehyde or haloacid) assays to provide initial values for each component. At the end of the reaction period (15 min in the presence of methoxyflurane and in the control without substrate and 10 min with benzphetamine), the reaction mixture was withdrawn from the oxygen chamber and aliquoted to measure ␣-ketoglutarate, hydrogen peroxide, and product.
Reaction Stoichiometry with Superoxide Determination-Superoxide was determined in the presence and absence of a NADPH-regenerating system. In both series of experiments, the normal sequence of addition of proteins and lipid was used and preincubation was for 1 h. The final concentration of P-450, reductase, and cytochrome b 5 (when present) was 0.2 M. Aliquots for hydrogen peroxide and product analysis were taken at 1.0 and again at 6.0 min. With the regenerating system, aliquots were also taken at 1.0 and 6.0 min for ␣-ketoglutarate analysis. Oxygen consumption and superoxide production were measured in separate parallel reaction mixtures. Superoxide was measured for 30 s 3 min after the start of the reaction.
End Point Conditions-In these experiments, NADPH was added to the reaction mixture to a final concentration of 0.15 mM, and the reaction was allowed to proceed until all of the NADPH was consumed (Gorsky et al., 1984). Reaction mixtures were prepared as described above for initial rate experiments with dilute cytochrome b 5 except that protein concentrations were higher. The final concentrations of assay components with no substrate or with either of the anesthetic agents as substrate were 1.0 M P-450, 0.5 M reductase, and 1.0 M cytochrome b 5 (when present). When the substrate was benzphetamine or dimethylaniline, final protein concentrations were 0.25 M for reductase and 0.5 M for P-450 and cytochrome b 5 . The reaction mixture was added to the oxygen chamber, and after the recording stabilized, NADPH was added to initiate the reaction. Upon completion of the reaction, as determined by the cessation of O 2 consumption, aliquots of the reaction mixture were removed for determination of the products. The depletion of NADPH was confirmed by measurement of the absorbance of the mixture at 340 nm.

Cytochrome b 5 Stimulates Equally Both the O-Demethylation and Dechlorination Pathways of Methoxyflurane Metabolism
In order to study the in vitro metabolism of methoxyflurane with purified enzymes and small reaction volumes it was necessary to develop a sensitive and specific assay for methoxydifluoroacetic and dichloroacetic acids, the products of the dechlorination and O-demethylation pathways of methoxyflurane metabolism, respectively (Fig. 1). Therefore, a selected ion monitoring gas chromatography, mass spectrometry (GCMS) assay was developed. Since methoxydifluoroacetic acid is not FIG. 1. Pathways of methoxyflurane metabolism. commercially available, it was synthesized. Trideuterated methoxydifluoroacetate was also synthesized for use as an internal standard in the assay. Aliquots of reaction mixture supernatants were analyzed for methoxydifluoroacetic and dichloroacetic acids after extraction of the free acids with ether and formation of methyl esters with diazomethane. Extraction recoveries for the procedure were 76% for methoxydifluoroacetic acid and 95% for dichloroacetic acid. Linear calibration curves were obtained with known dichloroacetic acid concentrations up to 6 M. At concentrations above 6 M, precise calibration required the use of standards that closely bracketed the sample concentrations due to the slight nonlinearity which results due to the presence of naturally occurring [ 13 C]dichloroacetic acid. Linear calibration was obtained in the analysis of methoxydifluoroacetic acid with concentrations from 0.04 M up to 100 M. The GCMS assays were able to detect 0.04 M dichloroacetic acid and 0.13 M methoxydifluoroacetic acid in a 100-l sample with a signal 3 times the standard deviation of the blanks.
Using the GCMS assays for the haloacid metabolites of methoxyflurane it was possible to demonstrate that cytochrome b 5 enhanced the metabolism by 5-7-fold at both the dichloromethyl carbon and the -OCH 3 group (Table I and Fig. 1). These results confirm the in vivo studies of methoxyflurane metabolism in rats and humans which have shown that the major route of metabolism is via the dechlorination pathway (Holaday et al., 1970;Selinsky et al., 1988aSelinsky et al., , 1988b. The Odemethylation pathway is the clinically relevant one because it gives rise to the renotoxic fluoride ion. It is common for a given substrate to undergo regiospecific oxidation at more than one carbon atom by a single isozyme of cytochrome P-450. However, without a structure of the active site of cytochrome P-450 2B4, one cannot determine whether it is the ease of abstraction of a hydrogen atom and/or the proximity of the substrate to the active oxygen species [FeO] 3ϩ within the substrate binding site that dictates which part of the molecule is preferentially oxidized. In experiments whose data are not shown, it was demonstrated that 2 mol of fluoride ion were formed for every mole of dichloroacetic acid produced. These results extend our previous demonstration of the production of 2 mol of fluoride ion for each mole of formaldehyde produced (Waskell and Gonzales, 1982). When a known amount of either dichloroacetic or methoxydifluoroacetic acid was added to the reconstituted system, neither compound was significantly degraded.

Under a Variety of Conditions, Cytochrome b 5 Always Increases the Efficiency but Not Necessarily the Absolute Rate of the Cytochrome P-450-catalyzed Reactions
The stoichiometry of methoxyflurane metabolism by purified cytochrome P-450 2B4 with and without cytochrome b 5 was compared to that with several other substrates under a variety of conditions in an attempt to assure the generality of the conclusions and perhaps gain insight into the cause of the variability of the results under different conditions. The results of these experiments are provided in Tables II-VI and are briefly summarized in the following discussion. Methoxyflurane is a relatively poor substrate for cytochrome P-450 2B4. In the absence of cytochrome b 5 , only 1-3% of the NADPH consumed was utilized for product, and even in the presence of cytochrome b 5 only 7-23% of the NADPH is utilized to degrade the anesthetic (Table VII). The remainder of the NADPH is utilized to reduce oxygen to the side products, superoxide, hydrogen peroxide, and presumably water. Water is assumed to be produced by the cytochrome P-450-catalyzed 4-electron reduction of oxygen since 2 mol of NADPH and 1 mol of oxygen are consumed which cannot be accounted for in any other way (Gorsky et al., 1984). The other substrates studied were benzphetamine and dimethylaniline, two relatively good substrates for P-450 2B4, and enflurane (CClFH-CF 2 -O-CHF 2 ), a clinically used volatile anesthetic. Enflurane, a pseudosubstrate, binds to the active site of cytochrome P-450 2B4 but is incapable of being oxidized by the enzyme. Addition of substrates to the reconstituted cytochrome P-450 monooxygenase system caused a 2-5-fold increase in the rate of NADPH and O 2 consumption, but had much less pronounced effects on the rate of H 2 O 2 production. The stimulation of NADPH consumption was least with enflurane (Х2-fold) and highest with benzphetamine and dimethylaniline (4 -5-fold).
Table VII summarizes the data from Tables II-VI and demonstrates that, under all reaction conditions and with all of the substrates tested, cytochrome b 5 acts to improve the efficiency of the reaction for product formation, i.e. more product is formed by the oxidation of a fixed amount of NADPH. The increase in metabolites was at the expense of superoxide, hydrogen peroxide, or water or some combination of these side products and varied with the reaction conditions and the substrate. With benzphetamine or dimethylaniline, this increased efficiency was sometimes but not always reflected in an increased rate of product formation and was even associated with a decreased rate of product formation in one instance (Table  VII). In contrast, the rate of methoxyflurane metabolism was stimulated by cytochrome b 5 under all of the conditions tested, although the stimulation observed ranged from 2-to 6.5-fold (Tables II and IV).
The second noteworthy feature of Table VII is the demonstration that cytochrome b 5 decreases the amount of NADPH consumed by 10 -60% under all experimental conditions except those described in Table II and section A of Table VII. In these experiments cytochrome b 5 was added as a very dilute solution (0.16 M) to the reconstituted reaction mixture.
It is also of interest that the amount of NADPH consumed by the turnover of cytochrome P-450 is approximately 40% greater during the metabolism of the good substrate benzphetamine than during the metabolism of the poorer substrate methoxyflurane under most reaction conditions (Table VII). This trend continues with the pseudosubstrate, enflurane, consuming less NADPH than methoxyflurane but more than cytochrome P-450 in the absence of substrate.

Comparison of Stoichiometry with Changes in Reaction Conditions
Effect of Addition of Cytochrome b 5 from a Dilute (0.16 M) versus a Concentrated (3.4 M) Solution-In these experiments the proteins were added in the "normal" sequence, cytochrome P-450, reductase, and finally cytochrome b 5 . Our results confirm the observations by Gorsky and Coon (1986) that the concentration of cytochrome b 5 at the time of its addition to the reconstituted system may influence the catalytic activity of the reconstituted P-450 system and alter the observed stoichiometry. The results in Table II were obtained when cytochrome b 5 was added as a dilute solution (0.16 M) to a mixture containing Although the conditions which were used to obtain the results for Table II would seem to disfavor the association of cytochrome b 5 with the other enzymes of the reconstituted system,   good stimulation of methoxyflurane metabolism was seen when dilute cytochrome b 5 was added. In fact, a slight stimulation of benzphetamine and dimethylaniline metabolism was also seen, in contrast to the results obtained when cytochrome b 5 was added from a concentrated stock solution. Also differing was the lack of inhibition of dilute cytochrome b 5 (Table II) on NADPH consumption compared to the uniformly inhibitory effect of concentrated cytochrome b 5 (Table III). Inverse Sequence of Protein Addition: 1) Cytochrome P-450, 2) Cytochrome b 5 , and 3) Reductase-The stoichiometry with methoxyflurane and benzphetamine as substrates was also determined using an "inverse addition" sequence (Table IVB) (Gorsky and Coon, 1986). That is, cytochrome b 5 was added to the cytochrome P-450 prior to the addition of cytochrome P-450 reductase. When cytochrome b 5 was added prior to reductase, NADPH consumption by the reconstituted system was greatly reduced. In addition, metabolism of benzphetamine to formaldehyde was inhibited (by 38%), while the metabolism of methoxyflurane was only stimulated 2-fold (Table IV). This series of experiments is key to understanding the effect of cytochrome b 5 on the mixed function oxidase system for two reasons. The first is that it demonstrates that cytochrome b 5 can markedly inhibit the overall activity of the system. The second is that it compares the metabolism of cytochrome b 5 -requiring and nonrequiring substrates under conditions where the overall reaction, i.e. NADPH consumption, is inhibited by cytochrome b 5 but the efficiency of the reaction increases.
Stoichiometry of Cytochrome P-450-catalyzed Reactions with a NADPH-regenerating System-It was of interest to determine the effect of cytochrome b 5 on the reconstituted system in the presence of a NADPH generating system, since in the majority of instances the activity of cytochrome P-450 is quantitated in the presence of an NADPH generating system, and it was not known whether the reaction stoichiometry would be different in the presence of the continuously high NADPH concentration that a regenerating system provides. NADPH consumption was measured in these experiments by quantitating the amount of ␣-ketoglutarate produced using a spectrophotometric assay (Friedmann, 1957). At a concentration of 30 M the assay gives acceptable precision with a standard deviation of 15% which improved to 3% at 350 M ␣-ketoglutarate. The results obtained using the regenerating system are provided in Table VB. They indicate that total NADPH consumption is increased in the presence of a regenerating system, but otherwise the results do not differ significantly from the results observed in the presence of decreasing amounts of NADPH, i.e. under initial rate (Tables II and VA) or end point conditions (Table VI). These findings indicate that the reaction stoichiometry is not influenced by the small change in NADPH concentration observed under initial rate conditions and suggest that the conclusions from stoichiometry experiments performed under initial rate and end point conditions may be extrapolated to  the more usual experimental conditions in which a regenerating system is used. Due to the fact that the amount of ␣-ketoglutarate produced in the absence of substrate is below the limit of detection, this control, which is not expected to be different from the numerous other controls, could not be performed (Table VB). Only the E. coli isocitrate dehydrogenase which had been purified for crystallography (Hurley et al., 1989) could be used in the experiments with the NADPHgenerating system. If the commercial non-manganese-containing pig heart isocitrate dehydrogenase was used, superoxide reacted with a nondialyzable component of the enzyme preparation (presumably isocitrate dehydrogenase) and generated extra water. A 5 M solution of ferrous ion could mimic the alterations in the stoichiometry of the cytochrome P-450-catalyzed reaction observed in the pig heart isocitrate dehydrogenase and generate extra water. Consequently, great care was taken in all of the reported experiments to ensure that the ferrous ion concentration was decreased to levels where it could not interfere and that a significant amount of superoxide was not consumed by components of the reaction mixture, thereby producing confounding side products and inaccurate results.
End Point Conditions-The results for reactions conducted under end point conditions are provided in Table VI. End point stoichiometry experiments have the advantage that they are easy to carry out and generally provide the most reproducible results. However, because it is the amount of NADPH which is added rather than reaction rates which determine the extent of the reaction, the results from the end point experiments cannot be compared directly with the initial rate experiments. A comparison of the relative efficiency of NADPH consumption for product formation can be made, and this has been provided in Table VII. Compared to the initial rate experiments, experiments conducted under end point conditions produce less hydrogen peroxide and more water. The reaction stoichiometry was the same in the presence and absence of superoxide dismutase, suggesting that superoxide was not reacting with other components of the reaction mixture.

Superoxide Is the Major Source of Hydrogen Peroxide in the Cytochrome P-450 2B4-reconstituted System
Measurement of the rate of autooxidation of ferrous oxy cytochrome P-450 2B4 in the presence of the different substrates and cytochrome b 5 provides information about the origin of the hydrogen peroxide generated in the reconstituted system. Previous studies have generally, although not universally, indicated that superoxide is the main source of the hydrogen peroxide produced by cytochrome P-450 (Ingelman-Sundberg and Johansson, 1980;Kuthan et al., 1978). Whether the hydrogen peroxide comes from dismutation of superoxide or dissociation of the 2-electron-reduced oxygen species directly from cytochrome P-450 has implications for the mechanism by which cytochrome b 5 increased the efficiency of the reaction.
In our experiments superoxide was quantitated by assessing its ability to reduce succinoylated cytochrome c. Succinoylated cytochrome c was used rather than cytochrome c because Х40% of its amino groups have been acylated, thereby inhibiting binding to its redox partners. As a result the succinoylated cytochrome c is reduced Х1000-fold slower by cytochrome b 5 and cytochrome P-450 reductase relative to unmodified cytochrome c, but only 3-10-fold slower by superoxide itself. The rate of reduction of succinoylated cytochrome c which could not

under a variety of reaction conditions
The results are presented as the percentage of NADPH utilized in the presence of the indicated substrate to produce product and H 2 O 2 . The numbers in parentheses are the reducing equivalents assumed to yield water even though the ratio of excess NADPH to oxygen consumption differs significantly from the expected ratio of 2.0. The data for sections A-F are calculated from Tables II, III, IVB, V, VB, and VI, respectively.   No substrate  Methoxyflurane  Enflurane Benzphetamine N,N-Dimethylaniline Ϫb 5 ϩb 5 Ϫb 5 ϩb 5 Ϫb 5 ϩb 5 Ϫb 5 ϩb 5 Ϫb 5 ϩb 5 be inhibited by superoxide dismutase was assumed to be the rate at which cytochrome b 5 and reductase directly reduced the modified cytochrome c. The reduction of succinoylated cytochrome c not inhibited by superoxide dismutase ranged from 4.5 to 70% of the total reduction rate. This value was subtracted from the final rate of superoxide production in Table V. Additional complications in accurately quantitating the amount of superoxide produced by autooxidation of cytochrome P-450 in a reconstituted system are: 1) the facile reaction of superoxide with itself and other reaction components and 2) the fact that cytochrome P-450 is not the only source of superoxide. The reactivity of superoxide was dealt with by deriving Equation 23, as described under "Appendix," which allows us to calculate the actual amount of superoxide produced from the experimentally determined values. The method described herein and the method previously described by Kuthan et al. (1982) for quantitating superoxide are similar except that the present solution is more general. Equation 23 under "Appendix" accounts for the reaction of superoxide with components of the assay mixture while Equation 14 in the report by Kuthan et al. (1982) has been simplified and assumes this does not occur. In most instances this is a good assumption. However, it should be confirmed experimentally. Equation 23 also allows a more accurate calculation of V max over a wider range of experimental conditions. For example, a double reciprocal plot of the experimentally observed rate of reduction of succinoylated cytochrome c versus cytochrome c concentration will lead to an overestimate of V max because a double reciprocal plot of Equation 14 is not a straight line at high cytochrome c concentrations.
Under the typical reaction conditions cytochrome P-450 reductase also simultaneously autooxidizes to superoxide at a measurable rate (4.7 nmol of superoxide produced/min/nmol of reductase/2 nmol of NADPH). Fortunately, the autooxidation of cytochrome b 5 is an order of magnitude slower. Since it is not known how the spontaneous rate of autooxidation of reductase in the complete reconstituted system is affected by the presence of cytochrome P-450, the values for superoxide in Table V have not been corrected for reductase autooxidation.
When the succinoylated cytochrome c assay was applied to the measurement of superoxide by the reconstituted system, a further complication was found. It could be demonstrated that in the first minute versus the following 5 min there was increased NADPH consumption and an overproduction of superoxide and presumably hydrogen peroxide. Pompon (1987) also observed a transient overproduction of hydrogen peroxide and underproduction of product immediately following addition of NADPH under similar experimental conditions. The reason for this transient overproduction of superoxide is not known. Because of this transient alteration in the reaction stoichiometry, all measurements were made between 1 and 6 min, except for superoxide. The superoxide measurements were made for only 30 s in the middle of the 1-6-min time period to assure that the succinoylated cytochrome c concentration would not be significantly altered during the course of the measurement. The stoichiometry of the metabolism of methoxyflurane and benzphetamine in the presence and absence of cytochrome b 5 is presented in Table V. In Table VA only the initial concentration of NADPH was 0.3 mM. It was not regenerated during the course of the reaction and had declined to as low as 0.2 mM in some experiments when the reaction was terminated. In Table  VB an NADPH-generating system was used to maintain NADPH at a constant concentration of 0.3 mM. Although the total amount of NADPH consumed was greater in the presence of the regenerating system, the overall reaction stoichiometry is essentially the same. Cytochrome b 5 decreased the absolute and relative amount of superoxide production during total methoxyflurane and benzphetamine metabolism (Table VII). In the presence of cytochrome b 5 a greater fraction of the reducing equivalents was redirected to product formation. The majority of the hydrogen peroxide observed in Table V is derived from the dismutation of superoxide since the molar ratio of superoxide to hydrogen peroxide was 2. In some instances, however, the superoxide to hydrogen peroxide ratio was less than 2, suggesting that a small amount of the hydrogen peroxide can also arise by dissociation of the protonated 2-electron-reduced oxygen directly from cytochrome P-450. The amount of hydrogen peroxide produced by direct dissociation from cytochrome P-450 was greatest in the control reactions without substrate where product cannot be generated after introduction of the second electron. In summary, any explanation for the mechanism of action of cytochrome b 5 will have to explain how cytochrome b 5 decreases autooxidation of oxyferrous cytochrome P-450 and simultaneously increases product formation.

DISCUSSION
Although the reactivity and overall stoichiometry of the reactions catalyzed by the cytochrome P-450 reconstituted system varied with the experimental conditions, the effect of cytochrome b 5 was similar in all cases; addition of cytochrome b 5 to the reconstituted system in the presence of both methoxyflurane and benzphetamine resulted in improved efficiency of product formation at the expense of the side products, superoxide, hydrogen peroxide, and water. With methoxyflurane this was always accompanied by an increased rate of product formation, from 2-6-fold, while with benzphetamine and dimethylaniline rates were unchanged, higher, or 40% lower, depending on reaction conditions. In order to understand these findings it is necessary to examine the results in Tables IV and VII in detail. For a particular set of experimental conditions the increase in reaction efficiency is approximately equal with both methoxyflurane and benzphetamine. However, under different experimental conditions the increased coupling varies from 2 to 20% of the overall NADPH consumption. If cytochrome b 5 always increases the efficiency of oxidation, why then does not cytochrome b 5 always increase production formation? The reason is that under certain experimental conditions cytochrome b 5 has a second effect in the reconstituted system, i.e. it may also inhibit the activity of the system, as measured by NADPH consumption, by as much as 60% (Table VIIC). Thus the amount of product generated will be the net result of the stimulatory and inhibitory effects of cytochrome b 5 . For example, in Table VIIC, it was observed that inverse addition of cytochrome b 5 (addition of cytochrome b 5 before reductase) inhibited NADPH consumption during benzphetamine and methoxyflurane oxidation by 40 and 60%, respectively, but increased the overall efficiency of the reaction by only 2%. Evidently, methoxyflurane is such a slowly metabolized, poorly coupled substrate that only 0.5% of the 59 nmol of NADPH consumed/min/nmol of cytochrome P-450 in the absence of cytochrome b 5 (Table VIIC) results in product formation. As a result a mere 2% increase in efficiency of the moderately depressed reaction (i.e. 2.5% of the 23 nmol of NADPH consumed/ min/nmol of cytochrome P-450 in the presence of cytochrome b 5 ) generates a small albeit detectable increase in the absolute amount of the product. Paradoxically the absolute amount of benzphetamine metabolism decreases under the identical conditions because a 40% decrease of product formation and NADPH consumption during the metabolism of a highly coupled (Х50%) good substrate cannot be counterbalanced by a 2% increase in coupling of a markedly inhibited reaction. Table VIIA also provides the explanation of why cytochrome b 5 some-times stimulates benzphetamine metabolism. If cytochrome b 5 does not inhibit the overall turnover of cytochrome P-450 as measured by NADPH consumption and increases the efficiency as it invariably does, product formation will be increased. Cytochrome b 5 did not significantly increase NADPH consumption in any of the experiments.
How does addition of cytochrome b 5 before reductase inhibit the mixed function oxidase system? Gorsky and Coon (1986) also observed this phenomenon with cytochrome P-450 2B4 and in addition demonstrated that inverse addition of a dilute solution of cytochrome b 5 was not inhibitory to the reaction. In fact, in the studies reported herein only those in which cytochrome b 5 was added as a dilute solution (Tables II and VII) was NADPH consumption not inhibited. Recent studies by Holloway and co-workers (Tretyachenko-Ladokhina et al., 1993) suggest a possible explanation for the different effects of cytochrome b 5 from solutions of dissimilar concentration. These investigators show that cytochrome b 5 self-associates into an octameric micellar structure at Х0.5 M, depending on the pH and ionic strength of the buffer. Thus cytochrome b 5 added from a 0.16 M dilute solution may have been monomeric, while the cytochrome b 5 in the 3.4 M concentrated solution was octameric. The 40 -60% inhibition of NADPH consumption by addition of octameric cytochrome b 5 prior to the reductase and lesser amounts of inhibition when cytochrome b 5 is added after reductase suggests that aggregated but not monomeric cytochrome b 5 may be inhibiting the reduction of cytochrome P-450 by reductase. Consistent with this notion is the study by Tamburini and Schenkman (1987) which convincingly demonstrated that cytochrome b 5 and cytochrome P-450 reductase bind at separate, nonoverlapping sites on the cytochrome P-450 2B4 molecule.
The presumed extra water formed during catalysis by cytochrome P-450 is thought to arise from the 2-electron reduction of the activated oxygen [FeO] 3ϩ species of the enzyme (Gorsky et al., 1984;Loida and Sligar, 1993). Loida and Sligar (1993) hypothesize that the reactive oxygen species of cytochrome P-450 (which has been estimated to have an oxidation-reduction potential of 1.45-1.75 V) partitions between substrate hydroxylation and a presumed further 2-electron reduction to water (Macdonald et al., 1989). With cytochrome P-450 camphor the amount of extra water produced is primarily dependent on the proximity of the substrate to the putative iron-oxo species (Loida and Sligar, 1993). Extra water was always observed during methoxyflurane oxidation but observed only under some experimental conditions during benzphetamine metabolism. For example, with benzphetamine as the substrate, extra water formation varied from 5 to 39% of NADPH consumption without cytochrome b 5 and from 0 to 30% with cytochrome b 5 . This lack of reproducibility in the extent of extra water formation was also observed by Gorsky et al. (1984). There is the possibility that the extra water could arise from some unknown side reaction or a Haber-Weiss type reaction. In fact in experiments with the pig heart isocitrate dehydrogenase NADPH-generating system, evidence was obtained that a component of the pig heart dehydrogenase preparation reacted with superoxide and resulted in extra water formation. This result was of major concern, and extreme caution has been used in interpretation of the findings of extra water even though in the remaining series of experiments there was no evidence that such reactions occurred. To rule out extra water production via a Haber-Weiss type of reaction 1 M Fe 3ϩ was added to reaction mixtures, and the amount of hydrogen peroxide and superoxide was determined. Addition of 1 M Fe 3ϩ did not alter the levels of hydrogen peroxide or superoxide measured. A concerted effort was made to keep the level of ferric ions in our reaction mixtures below 1 M by using only water and reagents that were known to result in final solutions of the reaction mixture containing less than 1 M Fe 3ϩ . The Haber-Weiss reaction has previously been well characterized in a reconstituted system, and if it were significant, it should have been detected in our control reactions (Winston and Cederbaum, 1983). Furthermore, addition of superoxide dismutase to reaction mixtures did not alter the observed stoichiometry.
One possible explanation which has been suggested for the increased efficiency for product formation and decreased rate of superoxide formation with the addition of cytochrome b 5 is that the introduction of the second electron is faster in the presence of cytochrome b 5 , resulting in more product formation at the expense of superoxide (Gorsky and Coon, 1986;Ingelman-Sundberg and Johansson, 1980). Experiments are currently in progress to directly examine the relative rates of introduction of the second electron by cytochrome b 5 and cytochrome P-450 reductase. This explanation also predicts that extra water production which is hypothesized to compete with substrate oxidation for the active oxygen species of cytochrome P-450 should also be increased by cytochrome b 5 . In fact, a decreased rate of water formation was calculated under most reaction conditions with methoxyflurane and benzphetamine. Previously it has been shown that, in order for cytochrome b 5 to support hydroxylation of substrates by cytochrome P-450, it must be capable of donating electrons to cytochrome P-450 (Canova-Davis et al., 1985;Morgan and Coon, 1984).
A second mechanism whereby cytochrome b 5 may increase reaction efficiency is that cytochrome b 5 may be causing a conformational change in cytochrome P-450 which stabilizes the binding of oxygen to cytochrome P-450 and/or inhibits protonation of the superoxide anion. This would decrease superoxide production and increase the concentration of the oxyferrous cytochrome P-450 intermediate which can proceed to product formation.
At present the autooxidation of cytochrome P-450 is thought to occur by a process formally identical to the autooxidation of myoglobin (Brantley et al., 1993;Cameron et al., 1993). In myoglobin, oxygen binding to the ferrous heme is markedly stabilized by hydrogen bonding to the N ⑀ -H of histidine 64. Autooxidation is rapid in mutants of myoglobin where His-64 is replaced by a hydrophobic amino acid incapable of hydrogen bonding to the liganded oxygen. Low pH also enhances the rate of autooxidation. This is hypothesized to be due to protonation of the ferrous oxy complex which then rapidly dissociates to the ferric protein and the protonated superoxide radical, HO 2 . .
Unprotonated anionic superoxide does not readily dissociate from a ferrous oxy heme protein. Recent studies of cytochrome P-450 suggest that oxygen may be stabilized by hydrogen bonding to the hydroxyl group of the conserved Thr-252 of cytochrome P-450 camphor and/or a water molecule buried in a nearby groove in the I helix (Gerber and Sligar, 1994;Imai et al., 1989;Martinis et al., 1989;Raag and Poulos, 1989;Ravichandran et al., 1993). By analogy with cytochrome P-450 camphor, cytochrome b 5 may be enhancing the binding between oxygen and Thr-302 in cytochrome P-450 2B4 (Nelson and Strobel, 1988). A third proposal is that the binding of cytochrome b 5 , which is known to cause a type I difference spectrum and decrease the K m of benzphetamine for cytochrome P-450 2B4 (Bonfils et al., 1981;Morgan and Coon, 1984;Tamburini and Schenkman, 1987), also alters the conformation of cytochrome P-450 such that the substrates methoxyflurane and benzphetamine are pushed closer to the reactive oxygen species and hence are more rapidly oxidized. Several investigators (Collins and Loew, 1988;Loida and Sligar, 1993;White et al., 1984White et al., , 1986 have demonstrated that the proximity of the substrate to the reactive oxygen species of cytochrome P-450 is a major determinant of its susceptibility to oxidation. Since the oxidation of both carbons of methoxyflurane are equally enhanced by cytochrome b 5 , it is possible that both carbons are moved closer to the oxidizing species of cytochrome P-450 in the cytochrome P-450cytochrome b 5 complex. It has also been reported that cytochrome b 5 markedly stimulates benzo(a)pyrene and lauric acid metabolism by cytochrome P-450 2B4 without any significant changes in the regiospecificity of the oxidation of these substrates (Brü nstrom and Ingelman-Sundberg, 1980;Morgan and Coon, 1984;Okita et al., 1981). On the other hand, cytochrome b 5 does change the regiospecificity of the metabolism of testosterone and 4-androstene-3,17-dione by cytochrome P-450 2B4. This could be explained if cytochrome b 5 caused one position of the substrate but not the other to be closer to the reactive oxygen species (Morgan and Coon, 1984). However, until additional structural data about the active site of cytochrome P-450 2B4 are available, direct experimental evidence for this proposal will be lacking. Although cytochrome b 5 is not known, to these authors' knowledge, to consistently decrease the efficiency of hydroxylation of a substrate, this hypothesis suggests that such a substrate might exist. That is, upon binding, cytochrome b 5 might alter the conformation of the active site of cytochrome P-450 in such a way that it pushed the substrate away from the oxidizing species.
The notion that cytochrome b 5 may alter the structure of cytochrome P-450 2B4 is supported by studies which have established the flexibility of cytochrome P-450 2B4 (Schwarz et al., 1984) and cytochrome P-450 camphor in the proximity of their active sites. The plasticity of the cytochrome P-450 camphor active site has been documented crystallographically. Inhibitors such as UK-67254-13, metyrapone, and phenylimidazole have been shown to alter the conformation of both the substrate access channel and the central region of the I helix which contains Thr-252 and forms part of the active site (Poulos and Howard, 1987;Raag et al., 1993).
If cytochrome b 5 is enhancing substrate oxidation by changing the conformation of the active site (but not increasing the binding between oxygen and Thr-252), how is it able to simultaneously decrease superoxide production? One possibility is that cytochrome b 5 itself or the conformational change it induces in cytochrome P-450 may decrease the access of protons to the oxy-ferrous complex. A second option is that the dissociation of the neutral superoxide radical from the active site may be sterically inhibited: 1) by cytochrome b 5 , 2) by the conformational change induced by cytochrome b 5 , and/or 3) by the substrate which may have been pushed closer to the hemebound oxygen. For example, camphor is known to inhibit by two orders of magnitude the rate of autooxidation of cytochrome P-450 camphor by sterically obstructing the dissociation of superoxide (Martinis et al., 1989).
The hypotheses for the mechanism of action of cytochrome b 5 discussed in the preceding paragraphs are not mutually exclusive. Cytochrome b 5 could both introduce the second electron at a faster rate than does cytochrome P-450 reductase and cause a conformational change in cytochrome P-450 which results in enhanced substrate oxidation and/or decreased autooxidation. Studies are currently underway to explore some of these issues.