(Received for publication, June 26, 1995)
From the
The complete stoichiometry of the metabolism of the cytochrome b (cyt b
)-requiring
substrate, methoxyflurane, by purified cytochrome P-450 2B4 was
compared to that of another substrate, benzphetamine, which does not
require cyt b
for its metabolism. Cyt b
invariably improved the efficiency of product
formation. That is, in the presence of cyt b
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 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
. Under some
experimental conditions cyt b
inhibits total NADPH
consumption. Whether stimulation, inhibition, or no change in product
formation is observed in the presence of cyt b
depends on the net effect of the stimulatory and inhibitory
effects of cyt b
. When total NADPH consumption is
inhibited by cyt b
, the rapidly metabolized,
highly coupled (
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 (
0.5-3%)
substrate, methoxyflurane, inhibition of total NADPH consumption by cyt b
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
(CHClCF
OCH
) 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.
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.
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
C
H
O
F
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
[
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.
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.
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.
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.
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.
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.
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.
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.
Reduction of succinoylated cytochrome c by superoxide occurs according to the following equation:
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
10
M
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:
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:
At steady state the rate of superoxide generation equals the rate of destruction:
Rearrangement and substitution from and gives:
Taking the square root and solving for v: