(Received for publication, August 14, 1995)
From the
The hydroxylation of (1R)-camphor by cytochrome
P450-CAM involves almost complete coupling of electron to oxygen
transfer. Modifications at C-5 of camphor, the normal site of
hydroxylation by P450-CAM, lead to as much as 98% uncoupling of
electron and oxygen transfer as well as to decreases in the rate of
electron uptake (up to 10-fold) and the rate of oxygenated product
formation (up to 210-fold). Two modes of uncoupling are seen: (a) two-electron uncoupling in which the decrease in
oxygenated product formation is balanced by increases in
HO
formation and (b) four-electron
``oxidase'' uncoupling where the NADH/O
ratio has
changed from one to nearly two and relatively little
H
O
is formed. Both enantiomers of
5-methylenylcamphor are two-electron uncouplers, while (1R)-
and (1S)-5,5-difluorocamphor and
(1R)-9,9,9-d
-5,5-difluorocamphor are
four-electron uncouplers. An intermolecular isotope effect of
11.7 is observed for oxygenation of C-9 in
(1R)-5,5-difluorocamphor. With this substrate, the significant
decrease in the rate of oxygenated product formation combined with the
large isotope effect suggest that the rate-limiting step has switched
from electron to oxygen transfer.
The cytochromes P450 (P450) ()are a family of b-type heme monooxygenases that catalyze the activation of
dioxygen for incorporation into a variety of organic
substrates(1, 2, 3, 4, 5, 6, 7) .
Much of our present knowledge of the structure and mechanism of P450
enzymes comes from investigations of the bacterial P450-CAM isolated
from Pseudomonas putida. P450-CAM catalyzes the highly
stereoselective hydroxylation of its physiological substrate,
(1R)-camphor, to form
(1R)-5-exo-hydroxycamphor as the sole product (Fig. R1). In addition, the enzyme will hydroxylate related bi-
and tricyclic molecules such as
norcamphor(8, 9, 10) ,
thiocamphor(11) , adamantane(12) ,
adamantanone(12) , camphane(11) , and
5,5-difluorocamphor (Fig. R1) (13) as well as epoxidize
5,6-dehydrocamphor (14) and 5-methylenylcamphor (Fig. R1)(15) . Both enantiomers of camphor and
5-methylenylcamphor are oxygenated by the enzyme(15) .
Recently, it has been found to be capable of hydroxylating and
epoxidizing a variety of molecules that bear no structural resemblance
to camphor such as ethylbenzene(16) , nicotine(17) ,
4-methyl-1-tetralone(18) , styrene(19) ,
1,1,1-trichloroethane(20) , thioanisole(21) , and
several others(22) .
Figure R1: Reactions 1-3.
Four well characterized intermediates (1-4, Fig. 1) have been observed in the reaction
cycle of
P450(1, 2, 3, 4, 5, 6, 7) .
The low-spin ferric resting state (1) becomes high-spin (2) upon substrate binding. Electron transfer from NADH via
two-electron transfer proteins, putidaredoxin reductase (PdR) and
putidaredoxin (Pd), yields the high-spin deoxyferrous state (3)
that is capable of binding O to generate the oxyferrous
state (4) or CO to produce the carbonmonoxy ferrous inhibitor
adduct (5). Addition of the second electron, the rate-limiting
step in the cycle(23) , is proposed to yield a ferric peroxide
adduct (6a) which can be protonated on oxygen to give a
hydroperoxide complex (6b). Protonation of the oxygen that
already bears a proton and heterolytic cleavage of the peroxide O-O
bond releases water and generates the proposed oxo-ferryl porphyrin
radical intermediate 7. Intermediate 7 then transfers an
oxygen atom to the hydrocarbon substrate, presumably by an oxygen
rebound mechanism(24) , to give the alcohol product and the
P450 state 1. With (1R)-camphor, the cycle just
described proceeds with nearly complete coupling (9, 15) of electron transfer (2
3 and 4
6) to oxygen transfer (7
1). Two modes of uncoupling electron transfer from oxygen
transfer after addition of the second electron have been
described(8, 16, 25) . Protonation of 6b on the unprotonated oxygen would lead to release of hydrogen
peroxide generating 2 and no oxygenated product (Path
A, 6b
2, Fig. 1, two-electron uncoupling).
Alternatively, reduction of 7 by two electrons and concurrent
protonation would give a second molecule of water (the
``oxidase'' reaction), regenerate 2 and produce no oxygenated product (Path B, 7
2, Fig. 1, four-electron oxidase uncoupling).
Figure 1:
Reaction cycle of cytochrome P450. RH represents the substrate (camphor or camphor analog) and R(O)H represents the oxygenated (hydroxylated or epoxidized)
product. The dianionic porphyrin macrocycle is abbreviated as a
parallelogram with nitrogens at the corners. Oxy-P450 (4) is shown as a
complex of ferric porphyrin and superoxide anion, but could also be
described as an adduct of neutral dioxygen and ferrous porphyrin.
States 6a, 6b, and 7 are hypothetical intermediates whose structures
have not been established. Structures 1, 2, and 7 are neutral (the dot and the positive charge on 7 indicate the radical state
and electron deficiency of the -electron system of the porphyrin
ring), while the overall charge on structures 3, 4, 5, and 6b is
-1 and on intermediate 6a is -2. Two modes of uncoupling
electron and oxygen transfer, A and B, are shown. See
text for further discussion of the events in the cycle and the nature
of the numbered intermediates.
In an effort to
probe the mechanism of dioxygen activation by P450-CAM through blockage
of the normal site of reaction, we have synthesized camphor analogues
with two fluorine atoms or an exocyclic olefin functionality at C-5. We
have previously reported that 5,5-difluorocamphor is hydroxylated at
C-9 to give the hydroxymethyl product (13) and that
5-methylenylcamphor is epoxidized to give the exo-epoxide
product(15) . Herein we describe in detail the substrate
binding constants along with the electron, dioxygen, and substrate
stoichiometries for the reactions of both (1R)- and
(1S)-enantiomers of the difluoro and methylenyl substrates as
well as for
(1R)-9,9,9-d-5,5-difluorocamphor.
Parallel studies have been carried out with both (1R)- and
(1S)-camphor. The difluoro and methylenyl substrates are
oxygenated at appreciably slower rates than (1R)-camphor. This
may reflect a change in the rate-limiting step from the second electron
transfer to a later step, possibly oxygen transfer. In addition, oxygen
transfer to the camphor analogues is uncoupled from electron transfer
to such an extent that in many cases, the majority of the electrons
that flow into the system do not participate in the production of
oxygenated product. Finally, the two types of substrates are uncoupled
in different ways: the difluoro substrates are four-electron uncouplers
while the methylenyl substrates are two-electron uncouplers.
Figure 2: Electronic absorption spectrum of (1R)-camphor bound (solid) and (1S)-camphor bound (dashed) cytochrome P450-CAM. Protein concentrations were 70 and 40 µM, respectively. Spectra were recorded at 4 °C in 20 mM phosphate buffer, pH 7.4, containing 100 mM KCl and under conditions of saturating substrate concentration (1 mM).
In analyzing the
crystal structures of both camphor-bound and camphor-free P450-CAM,
Poulos and co-workers (31, 32, 33) have
suggested that substrate specificity is achieved by specific
interactions between the substrate, (1R)-camphor, and
appropriately positioned amino acids in the substrate binding pocket.
The most important interactions are a hydrogen bond between
Tyr and the carbonyl oxygen on C-2 of camphor and van der
Waals contacts between Val
and the 8,9-geminal dimethyl
groups of camphor and between Leu
-Val
and
the 10-methyl group of camphor. Like (1R)-camphor,
(1S)-camphor reacts exclusively at the
5-exo-position. This suggests that the binding position of
(1S)-camphor may still be oriented by the hydrogen bond
between its carbonyl group and Tyr
. To maintain this
hydrogen bond and also position C-5 in the same relative position as in
(1R)-camphor requires that the protons on C-3 in
(1S)-camphor occupy the position where the 10-methyl group was
located in (1R)-camphor (Fig. S1). This modifies the
protein-substrate van der Waals interactions; the incomplete spin shift
upon binding (1S)-camphor indicates that the binding
interaction is not optimal. Nonetheless, hydroxylation of
(1S)-camphor still occurs at the exo position of C-5.
The same arguments regarding regio- and stereochemistry would seem to
apply to the epoxidation of (1R)- and
(1S)-5-methylenylcamphor. Interestingly, a recent study of the
hydroxylation of (1R)- and (1S)-norcamphor showed
that while C-5 is the primary site of hydroxylation for the
(1R)-enantiomer, C-6 was preferred for
(1S)-norcamphor(9) .
Figure S1: Scheme 1.
For 5,5-difluorocamphor, blocking C-5 with fluorines shuts off reaction at that carbon completely. The major product in the 1R case is the 9-alcohol. This is not surprising since C-9 is the second closest carbon to the heme iron after C-5(32, 36) . For the (1S)-enantiomer, the non-optimal binding and slow rate of oxygen transfer (Table 3) lead to multiple reaction sites including the only example of C-3 hydroxylation observed herein (Table 2).
With
(1R)-9,9,9-d-5,5-difluorocamphor, the
rate of NADH oxidation (557 min
) is actually higher
than the rate for the non-deuterated substrate (420
min
) (Table 1). Similar apparent inverse
isotope effects have been reported for norcamphor by Sligar and
co-workers(8) . Once again, however, it is important to
remember that with uncoupled substrates, the NADH oxidation rate
reflects not only diminished oxygenated product formation but also two-
and four-electron uncoupling. From the uncoupling and stoichiometry
data in Table 1, it is possible to calculate the rates of
formation of oxygenated products (Table 3). This analysis shows
that the 9,9,9-d
-substrate produces oxygenated
products at a much slower rate than undeuterated 5,5-difluorocamphor.
The apparent inverse isotope effect for NADH oxidation rates for the
9,9,9-d
-substrate clearly results from the
significantly increased amount of four-electron uncoupling observed
with that substrate. The data in Table 3also reveal that the
rate of oxygenated product formation can be decreased by as much as
210-fold upon modification of the substrate.
To factor out the
effects of four-electron uncoupling, it is convenient to define an
O turnover number by dividing the NADH oxidation rate by
the NADH/O
consumption ratio (Table 1). This provides
a turnover number based on O
consumption. With one
O
consumed per cycle of the enzyme, this value represents
the number of cycles the enzyme carries out per minute. From these
data, it is seen that d
- and d
-(1R)-difluorocamphor each turn over
about 10-fold more slowly than (1R)-camphor.
With 5,5-difluorocamphor, the
significant slowdown in the rate of formation of oxygenated products (Table 3) suggests that the rate-limiting step in the P450 cycle
may have changed from electron transfer to oxygen transfer (which in
turn is initiated by hydrogen atom abstraction). This conclusion is
strengthened by the intermolecular isotope effect of 11.7 for
hydroxylation of C-9 in 5,5-difluorocamphor (Table 4). With
5-exo- and 5-endo-deuterated camphors, Gelb et
al.(37) reported an intermolecular isotope effect of only
1.1-1.2 and concluded that oxygen transfer is sufficiently fast
that the intrinsic isotope effect, k/k
, for hydrogen atom
removal is masked. The intermolecular isotope effect of 11.7
for oxygenation of C-9 in 5,5-difluorocamphor matches the intramolecular values of 11 or greater reported by Hjelmeland et al.(38) and Groves et al.(39) for aliphatic hydroxylation by liver microsomal P450.
The fact that the intermolecular isotope effect is no longer masked
suggests that the rate of hydrogen atom abstraction, which initiates
oxygen atom transfer, has been slowed down sufficiently at C-9 in
5,5-difluorocamphor to replace the second electron transfer as the
rate-limiting step for oxygenation at that site.
With the three difluoro substrates, the
average amount of NADH oxidized is about 440 nmol/ml while the
consumption of dioxygen remains about 260 nmol/ml. The amount of
oxygenated product formed was also considerably lower, the system now
being 83-98% uncoupled. The NADH/O consumption ratio (Table 1) approaches 2.0 with increasing uncoupling. Relatively
little hydrogen peroxide is formed. These observations combined with
the apparent inverse deuterium isotope effect discussed earlier are
consistent with four-electron oxidase uncoupling for the
difluorocamphor substrates.
The exact causes for the uncoupling of
the P450 reaction cycle are not known. Extensive studies by Raag and
Poulos (33, 40) have failed to attribute it to any one
factor. Uncoupling may result from less complementary enzyme-substrate
fit or from the lack of the hydrogen bond between the substrate
carbonyl and Tyr since the hydroxylation of norcamphor and
camphane, respectively, by P450-CAM are substantially
uncoupled(11, 41) . Uncoupling is not related to the
fraction of low-spin ferric heme, since thiocamphor-bound P450-CAM and
the camphor-bound Tyr
-Phe P450-CAM mutant are only 65 and
59% high-spin, respectively, yet have a very high hydroxylation
efficiency (100 and 98%, respectively)(11) . An important
factor that promotes uncoupling seems to be the presence of extra
solvent water near the O-O bond undergoing cleavage. To avoid the two
modes of uncoupling requires the precise delivery of two protons
exclusively to the outer oxygen of the iron-bound peroxide to generate
the oxo-ferryl state (6a
6b
7) and
the prevention of electron/proton delivery to the oxo-ferryl state (to
avoid 7
2). Gerber and Sligar (42) have
proposed a distal charge relay system to accomplish the proper delivery
of protons to the iron-bound peroxide oxygen. The ``extra''
water in the active site during catalysis apparently provides an
alternate source of protons to iron-bound peroxide, 6a and 6b, and the oxo-ferryl intermediate, 7, promoting water
and/or hydrogen peroxide production. Removal of methyl groups from the
substrate (as in norcamphor) or in mutant P450-CAM enzymes where active
site valines are replaced with alanines (41) leads to
uncoupling. Substrates like ethylbenzene and
1,1,1-trichloroethane(16, 20) , which certainly do not
tightly fill the substrate binding pocket, also promote uncoupling. The
results reported herein with 5-methylenylcamphor suggest that addition
of a carbon atom to the camphor (the exocyclic olefin substituent at
C-5) also leads to uncoupling.
Uncoupling also occurs when the
potential substrate hydroxylation sites are blocked as has been
observed in the present study with 5,5-difluorocamphor (Table 1).
Ullrich and co-workers (43) examined the reaction of
perfluoro-n-hexane with liver microsomal P450 and saw no
product formation and a 2:1 NADPH/O consumption ratio, due
to the four-electron reduction of O
to H
O.
Similar results are reported in this study for 5,5-difluorocamphor
where there is very little formation of oxygenated product and a
NADH/O
consumption ratio approaching 2:1. Other
laboratories have previously reported a four-electron oxidase activity
for mammalian and bacterial P450s(8, 25) . Loida and
Sligar (16) have been able to vary the amount of two-electron
and four-electron uncoupling observed in the benzylic hydroxylation of
ethylbenzene through changes in the side chains of particular amino
acids in the substrate binding pocket. The results herein indicate that
the mode of uncoupling can be varied using different 5-substituted
camphor analogues.
Replacement of the hydrogens on C-9 of
5,5-difluorocamphor with deuteriums increases the extent of
four-electron uncoupling (Table 1). Analysis of the consumption
of NADH by 5,5-difluorocamphor and its d derivative under oxygen limiting conditions (Table 5)
reveals significant differences in the distribution of alcohol and
water products resulting from oxygen transfer from the reduction of
intermediate 7, respectively. Deuteration of C-9 diminishes the
formation of C-9 alcohol from 28.3 to 2.4% with a concomitant increase
in the water oxidation reaction from 69.3 to 95.2%. Clearly, the fate
of the oxo-oxygen atom in intermediate 7 is very sensitive to
the presence of deuteriums at C-9 in 5,5-difluorocamphor.
Interestingly, the extent of alcohol formation at C-6 is not affected
by deuteration at C-9.
In summary, the oxygenation of camphor
analogues in which the normal site of reaction, C-5, is either blocked
or modified occurs with decreases in the rate of NADH consumption and
with considerable uncoupling of electron and oxygen transfer.
(1R)-5-Methylenylcamphor has been shown to undergo
two-electron uncoupling wherein the decrease in oxygenated product is
compensated for by the production of HO
.
(1S)-5-Methylenylcamphor exhibits slow turnover but is only
slightly uncoupled. Both enantiomers of 5,5-difluorocamphor and
(1R)-9,9,9-d
-5,5-difluorocamphor are four-electron
oxidase uncouplers. They presumably still go through the oxo-ferryl
intermediate, 7 in Fig. 1, which is reduced to give a
second molecule of water. The slowdown in the rate of oxygenated
product formation (Table 3) and the intermolecular
isotope effect of greater than 11 for reaction at C-9 of
5,5-difluorocamphor (Table 4) suggests that the rate-limiting
step in the P450 cycle may have changed from electron transfer to
oxygen transfer with 5,5-difluorocamphor.