It has previously been demonstrated that the O-demethylation of the volatile anesthetic methoxyflurane (CHClCFOCH) by the major phenobarbital-inducible hepatic cytochrome P-450 2B4 of rabbit is markedly stimulated in the presence of cytochrome b (Canova-Davis et al., 1985; Canova-Davis and Waskell, 1984). Cytochrome b 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 while the metabolism of most other substrates, including benzphetamine, is minimally stimulated, not effected, or slightly inhibited in the presence of cytochrome b (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 :

4) transfer of a second electron from either cytochrome P-450 reductase or cytochrome b; 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;

6) cleavage of the oxygen-oxygen bond with formation of a high valency iron-oxo intermediate [Fe=O] 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 (); 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 , 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 () 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 (Gerber and 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 at 11 °C) than the introduction of the second electron (5 s) 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 oxy-cytochrome 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-electron-reduced 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 [Fe=O] (Atkins and Sligar, 1987; Martinis et al., 1989). In addition to providing protons, water in the substrate-binding 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 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 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.

EXPERIMENTAL PROCEDURES

Materials

()

Purification of Microsomal Enzymes

Analytical Methods

Determination of the Superoxide Radical Anion

The three unknowns, V, 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 µM, 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 and cytochrome P-450 reductase. The reaction was usually measured for 30 s at a cytochrome P-450 concentration of 0.2 µM 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 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 (using the Marquardt-Levenberg algorithm of Sigma Plot), allowing the parameters K, V, 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 was considered to be the true rate of superoxide generation in the reconstituted system. The V 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 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 µM Fe to the reaction mixture did not affect the amount of superoxide measured whereas concentrations of Fe greater than 1 µM decreased the amount of superoxide measured.

Measurement of Methoxydifluoroacetic and Dichloroacetic Acids

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 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 CHOFNa 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 [H]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 µg of [H]methoxydifluoroacetic acid and 0.4 µg of 2,2-[2-C]dichloroacetic acid in 5 µl of water, was added to a 100-200-µl 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 µl, 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 0.53-mm GS-Q column (J& 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-O-CF) were monitored at m/z 81.1 and 84.1 for the methyl esters of methoxydifluoroacetic acid and [H]methoxydifluoroacetic acid, respectively. The fragment ion CHCl 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

Assay of Hydrogen Peroxide

NADPH Consumption (Spectrophotometrically and by -Ketoglutarate Production)

Oxygen Consumption

Measurement of Fluoride Ion Levels

Determination of Non-heme Iron

Assay of Protein Concentration

General Procedures for Determining Reaction Stoichiometry

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 µl were taken for hydrogen peroxide analysis, and aliquots of 200 µl 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 g for 5 min), and the supernatant was assayed for haloacid metabolites of methoxyflurane, as described previously.

Individual Reaction Conditions

Initial Rate Conditions

Reactions with Inverse Addition of Cytochrome b

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 (when present) was 0.2 µM with methoxyflurane or without substrate and 0.1 µM 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

End Point Conditions

RESULTS

Cytochrome b Stimulates Equally Both the O-Demethylation and Dechlorination Pathways of Methoxyflurane Metabolism

Figure 1: Pathways of methoxyflurane metabolism.

Using the GCMS assays for the haloacid metabolites of methoxyflurane it was possible to demonstrate that cytochrome b enhanced the metabolism by 5-7-fold at both the dichloromethyl carbon and the -OCH group ( Table 1and 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., 1988a, 1988b). The O-demethylation 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] 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 Always Increases the Efficiency but Not Necessarily the Absolute Rate of the Cytochrome P-450-catalyzed Reactions

Table 7summarizes the data from Table 2Table 3Table 4Table 5Table 6and demonstrates that, under all reaction conditions and with all of the substrates tested, cytochrome b 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 7). In contrast, the rate of methoxyflurane metabolism was stimulated by cytochrome b under all of the conditions tested, although the stimulation observed ranged from 2- to 6.5-fold ( Table 2and Table 4).

The second noteworthy feature of Table 7is the demonstration that cytochrome b decreases the amount of NADPH consumed by 10-60% under all experimental conditions except those described in Table 2and section A of Table 7. In these experiments cytochrome b was added as a very dilute solution (0.16 µM) 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 7). 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 from a Dilute (0.16 µM) versus a Concentrated (3.4 µM) Solution

Inverse Sequence of Protein Addition: 1) Cytochrome P-450, 2) Cytochrome b, and 3) Reductase

Stoichiometry of Cytochrome P-450-catalyzed Reactions with a NADPH-regenerating System

End Point Conditions

Superoxide Is the Major Source of Hydrogen Peroxide in the Cytochrome P-450 2B4-reconstituted System

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 40% of its amino groups have been acylated, thereby inhibiting binding to its redox partners. As a result the succinoylated cytochrome c is reduced 1000-fold slower by cytochrome b 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 be inhibited by superoxide dismutase was assumed to be the rate at which cytochrome b 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 5. 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 , 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. under ``Appendix'' accounts for the reaction of superoxide with components of the assay mixture while 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. also allows a more accurate calculation of V 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 because a double reciprocal plot of 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 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 5have 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 is presented in Table 5. In Table 5A 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 5B 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 decreased the absolute and relative amount of superoxide production during total methoxyflurane and benzphetamine metabolism (Table 7). In the presence of cytochrome b a greater fraction of the reducing equivalents was redirected to product formation. The majority of the hydrogen peroxide observed in Table 5is 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 will have to explain how cytochrome b 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 was similar in all cases; addition of cytochrome b 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 Table 4and Table 7in 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 always increases the efficiency of oxidation, why then does not cytochrome b always increase production formation? The reason is that under certain experimental conditions cytochrome b 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 7C). Thus the amount of product generated will be the net result of the stimulatory and inhibitory effects of cytochrome b. For example, in Table 7C, it was observed that inverse addition of cytochrome b (addition of cytochrome b 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 (Table 7C) 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) 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 (50%) good substrate cannot be counterbalanced by a 2% increase in coupling of a markedly inhibited reaction. Table 7A also provides the explanation of why cytochrome b sometimes stimulates benzphetamine metabolism. If cytochrome b 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 did not significantly increase NADPH consumption in any of the experiments.

How does addition of cytochrome b 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 was not inhibitory to the reaction. In fact, in the studies reported herein only those in which cytochrome b was added as a dilute solution ( Table 2and Table 7) 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 from solutions of dissimilar concentration. These investigators show that cytochrome b self-associates into an octameric micellar structure at 0.5 µM, depending on the pH and ionic strength of the buffer. Thus cytochrome b added from a 0.16 µM dilute solution may have been monomeric, while the cytochrome b in the 3.4 µM concentrated solution was octameric. The 40-60% inhibition of NADPH consumption by addition of octameric cytochrome b prior to the reductase and lesser amounts of inhibition when cytochrome b is added after reductase suggests that aggregated but not monomeric cytochrome b 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 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] 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 and from 0 to 30% with cytochrome b. 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 µM Fe was added to reaction mixtures, and the amount of hydrogen peroxide and superoxide was determined. Addition of 1 µM Fe 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 µM by using only water and reagents that were known to result in final solutions of the reaction mixture containing less than 1 µM Fe. 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 is that the introduction of the second electron is faster in the presence of cytochrome b, 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 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. 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 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 may increase reaction efficiency is that cytochrome b 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. 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 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, which is known to cause a type I difference spectrum and decrease the K 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., 1984, 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, it is possible that both carbons are moved closer to the oxidizing species of cytochrome P-450 in the cytochrome P-450-cytochrome b complex. It has also been reported that cytochrome b 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 (Brünstrom and Ingelman-Sundberg, 1980; Morgan and Coon, 1984; Okita et al., 1981). On the other hand, cytochrome b 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 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 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 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 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 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 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, 2) by the conformational change induced by cytochrome b, and/or 3) by the substrate which may have been pushed closer to the heme-bound 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 discussed in the preceding paragraphs are not mutually exclusive. Cytochrome b 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.

APPENDIX

In the following analysis a steady state equation is derived which relates the actual rate of superoxide production to the experimentally observed rate of reduction of succinoylated cytochrome c. It accounts for the three reactions of superoxide in a reconstituted system: 1) dismutation to hydrogen peroxide, 2) consumption by reaction components, and 3) reduction of cytochrome c.

Definitions

Reduction of succinoylated cytochrome c by superoxide occurs according to the following equation:

Thus,

The measurements were performed for a short period of time (30 s) with an excess of succinoylated cytochrome c so that the [cyt c] in the above equation remained constant.

The dismutation of superoxide occurs according to the following equation:

where O is the conjugate base of a weak acid HO whose pK is 4.7 and HO is the conjugate base of hydrogen peroxide. At pH 7.4 the effective dismutation rate constant will be 2 10M s (Fridovich, 1989). The rate of dismutation which is second order in superoxide concentration is as follows:

where K is the equilibrium constant between the acid and conjugate base forms of superoxide.

At fixed pH, hydrogen ion concentration is constant and a new constant can be defined:

Thus,

The rate of the reaction of superoxide with reaction components is assumed to occur according to the following equation:

where [U] is the concentration of the unknown reactant. In our experiments we found that even when significant side reactions were occurring, the reduction of succinoylated cytochrome c was linear during the measurement period. This means that neither the succinoylated cytochrome c nor the unknown reactant U is significantly depleted during the measurement period. The concentration of the unknown reactant, U, was therefore assumed to be constant for a given set of experimental conditions. Thus can be simplified as follows:

and,

At steady state the rate of superoxide generation equals the rate of destruction:

Rearrangement and substitution from and gives:

Solving 5 for [O]:

Substituting into 14

Define new constants:

Substituting into :

Solving for v:

Completing the square:

Taking the square root and solving for v:

Simplifying:

FOOTNOTES

*

This research was supported in part by National Institutes of Health Grant GM35533 and a VA Merit Review Grant (to L. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

To whom correspondence and reprint requests should be addressed: Dept. of Anesthesia (129), University of California, VA Medical Center, 4150 Clement St., San Francisco, CA 94121. Tel.: 415-750-2069; Fax: 415-750-6946.

()

The abbreviations used are: DLPC, dilauroyl L-3-phosphatidyl choline; GC, gas chromatography; MS, mass spectrometry.

ACKNOWLEDGEMENTS

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