J. Am. Chem. Soc. 1987, 109, 3754-3760
`discussing the structure of the excited-state TEA-ethanol complex,
`Kohler proposed that the O atom is coordinated to the quasica-
`tionic N atom core of the Rydberg excited TEA molecule.15 Were
`this to be true, it fails to explain why a donor such as tetra-
`hydrofuran shows no evidence of complex formation with elec-
`tronically excited TEA. Likewise, intramolecular complexation
`in amino ethers III and IV is not observed.7 Thus only protic
`species (H20, D20, ROH) appear to form emissive stoichiometric
`complexes with electronically excited saturated tertiary amines.
`°
`
`^
`
`3754
`
`are symbolized as B and B*, respectively. The energy difference
`between the amine-water complex which is produced subsequent
`to photon emission (and which presumably represents the equi-
`librium structure of the excited amine-water complex) and the
`“normal” ground-state amine-water complex is symbolized by Ex.
`A Forster cycle analysis of these state energies indicates that
`£>a + £,„v = B* + Efc + Ex - B
`Since each of these terms can be measured (or estimated) except
`for Ex, a value of this quantity can be determined.
`It should be
`realized that Ex, ordinarily represented as a repulsion energy in
`exciplex and excimer systems (because the ground states of these
`species are dissociative), need not necessarily be very large. That
`is, the energy of the amine-water complex produced after photon
`to that of the ground-state
`emission may be nearly equal
`amine-water hydrate.
`On the basis of the fluorescence energies of free and water-
`complexed TEA, the binding energies of the ground and elec-
`tronically excited state TEA-water complexes, and the use of a
`value of 8 kcal/mol as the inversion barrier for TEA,26 Ex is
`estimated to be ca. 13 kcal/mol. Data for the other amines suggest
`similar values for Ex. This value seems rather large if one assumes
`that the structure of the excited-state TEA-H20 complex involves
`the water molecule with the O-H bond directed along the C3 axis
`of the (planar) amine, I. The 13 kcal assigned to Ex represents
`the energy of this structure (in which, however, excitation energy
`is lost) relative to the (ground-state) TEA-H20 hydrate II.
`In
`H"o
`H
`
`A
`
`I
`
`  ^~  /
`nr
`in
`In order to rationalize the relatively large repulsion energy
`assigned for the TEA-H20 system, we propose that a very dif-
`ferent bonding arrangement exists in the excited-state complex
`relative to the ground-state hydrate. One such possibility is the
`four-center structure, V, shown below. The electrostatic reorg-
`HxxO-H
`"-rM
` 
`anization of the amine in the Rydberg excited state results in a
`quasicationic N atom core which coordinates with the O atom
`in H20 (and presumably also in ethanol). Additional stability
`is achieved by the electrostatic attraction of the sterically unen-
`cumbered H atom with the quasianionic (nucleophilic) periphery
`of the amine, such as the C atoms. Model calculations using
`appropriately extended basis orbitals on structures such as V are
`needed to assess the reasonableness of this suggestion.
`Acknowledgment. The donors of the Petroleum Research Fund,
`administered by the American Chemical Society, are acknowl-
`edged for partial support of this research.
`
`n
`I
`(26) (a) Bushweller, C. H.; Fleischman, S. H.; Grady, G. L; McGoff, P.;
`Rithner, C. D.; Whalon, M. R.; Brennan, J. G.; Marcantonio, R. P.; Do-
`mingue, R. P. J. Am. Chem. Soc. 1982, 104, 6224.
`(b) Fleischman, S. H.;
`Bushweller, C. H. J. Comput. Chem. 1985, 6, 249.
`
`Metabolic Switching in Cytochrome P-450cam: Deuterium
`Isotope Effects on Regiospecificity and the
`Monooxygenase/Oxidase Ratio
`William M. Atkins and Stephen G. Sligar*
`Contribution from the Departments of Biochemistry and Chemistry, University of Illinois,
`Urbana, Illinois 61801. Received September 29, 1986
`
`Abstract: Cytochrome P-450cam, isolated and purified to homogeneity from the soil bacterium Pseudomonas pulida, has been
`shown to catalyze the hydroxylation of the substrate analogue norcamphor to form three distinct products and yields:
`5-exo-hydroxynorcamphor (45%), 6-exo-hydroxynorcamphor (47%), and 3-exo-hydroxynorcamphor (8%). Specific deuteriation
`of the norcamphor skeleton at the 5-, 6-, and 3-positions drastically alters this product distribution, indicating a substantial
`deuterium isotope effect. When the sum total of all oxygenated products formed in the presence of norcamphor is compared
`to the number of reducing equivalents consumed in the reaction (NADH), a striking unaccountability of electrons is observed.
`These are shown to reside in excess water produced by the four-electron reduction of atmospheric dioxygen by P-450cam. Metabolism
`of specifically deuteriated norcamphor demonstrates a deuterium isotope effect on the branching ratio of substrate hydroxylation
`to excess water production and suggests that this oxidase activity of P-450cam results from the two-electron reduction of a single
`oxygen-iron intermediate,
`[FeO]3+.
`
`Cytochrome P-450cam, an extensively characterized mono-
`oxygenase derived from Pseudomonas putida, catalyzes the regio-
`
`and stereospecific hydroxylation of the monoterpene camphor to
`In this tightly
`afford 5-exo-hydroxycamphor as the sole product.1
`
`0002-7863/87/1509-3754S01.50/0
`
`© 1987 American Chemical Society
`
`Downloaded via WASHINGTON STATE UNIV on April 7, 2020 at 20:42:14 (UTC).
`
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`
`Apotex Ex. 1025
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`IPR2021-01507
`
`

`

`Metabolic Switching in Cytochrome P-450cam
`coupled reaction cycle, two reducing equivalents originating in
`NADH are transferred sequentially to the cytochrome via pu-
`tidaredoxin reductase and putidaredoxin with the subsequent 0-0
`bond scission of molecular oxygen. Nearly all reducing equivalents
`are accounted for by substrate hydroxylation and monooxygenase
`derived water, and with minimal production of hydrogen peroxide.
`the cytochrome P-450 systems isolated from the
`In contrast,
`hepatic endoplasmic reticulum are much less tightly coupled both
`in vivo as well as in reconstituted reactions. Here, as much as
`half of the pyridine nucleotide supplied reducing equivalents are
`used to generate hydrogen peroxide. Recently, Gorsky et al.
`demonstrated an additional fate for the electrons and molecular
`oxygen with several of these hepatic cytochrome P-450’s.1 2 They
`demonstrated that these liver microsomal
`isozymes were also
`capable of performing an oxidase-type reduction of atmospheric
`dioxygen, requiring four electrons and presumably generating 2
`equiv of water. Evidence suggested that H202 was not an in-
`termediate in this oxidase activity. Others have also reported a
`2:1 stoichiometry of NADPH/Ó2 in various liver microsomal
`systems and have proposed direct reduction of 02 to H203·4 with
`the efficacy of this oxidase activity varying with isozyme and
`organic substrate. Although investigations have established the
`existence of a cytochrome oxidase activity in these cytochrome
`P-450 systems, the mechanism of input of the additional two
`reducing equivalents into the normal monooxygenase reaction cycle
`has remained undetermined.
`An independent focus of investigation within the P-450 fields
`has involved the use of deuterium isotope effects to characterize
`the intermediacy of a
`the mechanism of C-H bond cleavage,
`substrate radical, and the nature and identity of the hydrogen
`abstracting species. At least two types of metabolic switching have
`been observed upon substitution of deuterium at an oxidizable
`carbon. Gelb et al.5 found that both the 5-exo and the 5-endo
`hydrogens of the camphor skeleton were candidates for abstraction
`and that deuterium substitution at either position altered the
`exo/endo selectivity of hydrogen removal without affecting the
`rigid specificity of exo oxygen rebound. White et al.6 found that
`the stereospecificity of hydrogen abstraction as well as oxygen
`rebound was affected by deuterium substitution on phenylethane.
`A second type of metabolic switching, in which new metabolic
`products have been obtained, has also been observed. Harada et
`al.7 reported that with the hepatic phenobarbitol and 3-methyl-
`cholanthrene induced cytochromes P-450, placement of deuterium
`the  -carbon of 7-ethoxycoumarin results in formation of a
`at
`second metabolite in addition to the normal O-deethylated product.
`Similarly, Jones et al.8 recently described how this type of met-
`abolic switching could be used to determine an intrinsic isotope
`effect for hydroxylation of octane by the rat phenobarbitol induced
`isozyme P-450b. Also, Dawson and co-workers demonstrated the
`formation of 9-hydroxycamphor from 5,5-gem-difluorocamphor
`by P-450cam.10
`
`(1) Silgar, S. G.; Murray, R. I. Cytochrome P-450 Structure, Mechanism
`and Biochemistry, Ortiz de Montellano, P. R., Ed.; Plenum: New York, 1986;
`pp 443-479.
`(2) Gorsky, L. D.; Koop, D. R.; Coon, M. J. J. Biol. Chem. 1984, 259,
`6812.
`(3) Staudt, H.; Lichtenberger, F.; Ullrich, V. Eur. J. Biochem. 1974, 46,
`
`99.
`
`I. Biochem. Biophys. Res. Commun.
`
`(4) Zhukov, A. A.; Archakov, A.
`1982, 109, 813.
` . H.; Heimbrook, D. C.; Malkonen, P.; Silgar, S. G. Bio-
`(5) Gelb,
`chemistry 1982, 21, 370.
`(6) White, R. E.; Miller, J. P.; Faureau, L. V.; Bhattacharyya, A. J. Am.
`Chem. Soc. 1986, 108, 6024.
`(7) Harada, N.; Miwa, G. T.; Walsh, J. S.; Lu, A. Y. J. Biol. Chem. 1984,
`259, 3005.
`(8) Jones, J. P.; Korzekwa, K. R.; Rettie, A. E.; Trager, W. F. J. Am.
`Chem. Soc. 1986, 108, 7074.
`(9) Lipscomb, J. D.; Sligar, S. G.; Namtvedt, M. J.; Gunsalus, I. C. J. Biol.
`Chem. 1976, 251, 1116.
`(10) Eble, J. S.; Dawson, J. H. J. Biol. Chem. 1984, 259, 14389.
`
`J. Am. Chem. Soc., Vol. 109, No. 12, 1987
`
`3755
`
`Substrate :
`
`/Norcamphor
`
`BC
`
`Internal
`Standard
`
`J_I_1_I
`I
`0
`4
`6
`2
`8
`Rentention Time (minutes)
`
`Retention Time (minutes)
`Figure 1. Gas chromatogram of norcamphor metabolites. Enzyme
`turnovers and gas chromatography were performed as described in the
`Experimental Section. After completion of the reaction, and prior to
`extraction, a known amount of internal standard (3-fvnfo-bromocamphor)
`(A) metabolite profile obtained from the norcamphor sub-
`was added:
`strate; (B) metabolite profile obtained from 5,6-exo,exo-norcamphor-
`5,6-d2.
`In view of the efficient production of 5-exo-hydroxycamphor
`by the bacterial P-450cam and the tight coupling of this system,
`wherein there is efficient channeling of pyridine nucleotide derived
`reducing equivalents into monooxygenase stoichiometry, we have
`chosen this system to explore the metabolic fate of dioxygen and
`substrate under varying conditions. Herein we describe results
`obtained with cytochrome P-450cam in the presence of the substrate
`analogue norcamphor, which earlier experiments suggested to be
`unmetabolized by this system.9 Careful analysis shows, however,
`that P-450cam demonstrates a prolific four-electron oxidase activity
`as well as substrate hydroxylation and a significant degree of
`metabolic switching upon deuteriation of the norcamphor sub-
`is the demonstration of an
`strate. Most intriguing, however,
`isotopically sensitive branching which affects the mono-
`oxygenase/oxidase ratio and which offers compelling evidence for
`intermediate that is responsible for hydrogen abstraction
`a common
`and is also susceptible to a two-electron reduction to form water
`in an activity paralleling that of cytochrome oxidase.
`
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`

`3756
`
`J. Am. Chem. Soc., Vol. 109, No. 12, 1987
`
`Atkins and Sligar
`
`Table II. Stereochemistry of Norcamphor Hydroxylation"
`substrate/
`buffer
`I in H20
`
`du %
`da, %
`d2, %
`product
`5-exo-hydroxy 4.6 ± 0.6 94.4 ± 0.5 0.96 ± 0.09
`1.1 ± 0.2
`6-exo-hydroxy 2.8 ± 0.9 95.9 ± 0.9
`98.07
`5-exo-hydroxy 0
`1.93
`
`norcamphor in
`D20
`
`Figure 2. Electron-impact mass spectra (70 eV) of norcamphor metabolites:
`(A) 3-exo-hydroxynorcamphor; (B) 6-exo-hydroxynorcamphor; (C)
`5-exo-hydroxynorcamphor. The spectra shown in the right-hand panel are of the corresponding authentic compounds.
`Results
`Table I. Reglospecificity of Norcamphor Hydroxylation
`Hydroxylated Products Derived from Norcamphor. Three
`relative percent of product, %
`found to be produced from nor-
`hydroxylated products were
`6-exo
`substrate0
`3-exo
`5-exo
`camphor metabolism by cytochrome P-450cam. The formation of
`each is strictly dependent on the presence of NADH, P-450cam,
`47
`45
`norcamphor
`8
`I
`and the purified electron-transfer proteins putidaredoxin and
`52
`27
`21
`II
`49
`49
`N ADH-putidaredoxin reductase. These norcamphor metabolites
`2
`III
`44
`46
`10
`identified by comparison to authentic samples with gas
`were
`“The symbols used are I, 5,6,-exo,exo-norcamphor-.5,6-</2; II, nor-
`chromatography and mass spectral analysis. A typical GC
`camphor-.?,i-i?2; and III, 5,6-exo,exo-norcamphor-?,?,.5,6-i?4.
`chromatogram of the reaction products when norcamphor was
`used as substrate is shown in Figure 1 A. The corresponding 70-eV
`electron impact mass spectrum of the products (A, B, C) is shown
`in Figure 2.
`The 3-exo-hydroxynorcamphor (A) and 5-exo-
`hydroxynorcamphor (C) produced in the reaction are identical
`with synthetic standards obtained as described in the Experimental
`Section. The third metabolite (B) has been assigned as 6-exo-
`hydroxynorcamphor based on GC mass spectral analysis and the
`(1) Metabolite B is not 5-enrfo-hydroxynorcamphor.
`following:
`The 5-endo alcohol was also prepared as described in the Ex-
`perimental Section, and it is apparent from GC analysis that the
`5- endo isomer is not present in the reaction mixture. With the
`GC conditions used, 5-emfo-hydroxynorcamphor had a retention
`time of 5.9 min and was easily separated from any of the products
`(2) Production of metabolite B is sensitive to deuterium
`observed.
`substitution at the 6-position, and from selective ion monitoring
`mass spectral analysis, it is apparent that B undergoes nearly
`complete loss of one deuterium when 5,6-exo,cxo-norcamphor-
`5,6-d2 is the substrate. These results are presented in more detail
`in the following discussion of stereochemistry. By analogy to the
`situation observed for the 5-alcohol, it is reasonable to assume
`that the 6-alcohol that is formed with loss of 6-exo deuterium is
`It
`arrived at with a preponderance of retention of configuration.
`is unlikely that the 5-alcohol would be formed with nearly complete
`retention and the 6-alcohol would be formed with nearly complete
`inversion. This line of reasoning leads us to conclude that B is
`the fragmentation
`6- exo-hydroxynorcamphor.
`Furthermore,
`pattern in the mass spectrum of B is entirely consistent with
`is noteworthy that both of the
`It
`6-exo-hydroxynorcamphor.
`isomeric 7 - hydroxy norcamphors were synthesized and shown to
`be absent from the metabolite profile of norcamphor.
`the 5- and 6-carbons of
`When deuterium is substituted at
`norcamphor, there is a significant intramolecular isotope effect
`
`98.43
`1.59
`6-exo-hydroxy 0
`“Deuterium content of the products was determined by ion selective
`monitoring GCMS. Peak intensities for ions corresponding to m/z
`128, 127, 126, 125, and 124 were determined. For both the 5- and
`6-alcohols (undeuteriated), the relative intensity of M+ -
`less
`1 was
`than 6% of the M+.
`
`which results in a change in the relative and absolute yield of each
`product (Figure 1); no new products appear to be formed. When
`various combinations of deuterium are present on the substrate,
`the product profile changes in a corresponding fashion. These
`It is apparent that, in sharp
`results are summrized in Table I.
`to what has been observed with camphor as substrate,
`contrast
`there is a deuterium-dependent intramolecular isotope effect on
`the regioselectivity of hydroxylation. Altered regioselectivity of
`camphor hydroxylation is observed only when a large kinetic
`barrier to hydroxylation has been presented, as was demonstrated
`with 5,5 -gem-difluorocamphor metabolism to 9-hydroxycamphor.10
`Stereochemistry of Norcamphor Hydroxylation. The stereo-
`chemical course of norcamphor hydroxylation at the 5- and 6-
`positions was investigated by determining the deuterium content
`of the corresponding alcohols when 5,6-exo,exonorcamphor-5,6-if2
`was metabolized. The results are summarized in Table II. Several
`interesting features of norcamphor hydroxylation contrast
`the
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`

`Metabolic Switching in Cytochrome P-450cam
`
`Figure 3. Recovery of hydrogen peroxide in the presence of norcamphor.
`The H202 recovered after addition of varying amounts of exogenous
`H202 was quantitated as described in the Experimental Section. The
`plotted values are corrected for the amount which is produced in the
`absence of added H202 (88 nmol). The dashed line represents the the-
`oretical yield of 100% recovery.
`
`stereochemical course observed with camphor.5 It is apparent that
`the 5-alcohol obtained from norcamphor results from nearly
`complete specificity for exo deuterium abstraction, with only minor
`It is likely
`endo abstraction followed by inversion of configuration.
`that due to the primary isotope effect, this stereochemical result
`significantly underestimates the exo specificity that occurs when
`protium is present at both the exo and endo face. A portion of
`the d2 product may simply arise from a trace amount of substrate
`with label at the endo position. Gelb et al.5 found, in studying
`the metabolism of the normal substrate camphor,
`that with
`deuterium present at the endo position there was still approxi-
`mately 20% endo abstraction with subsequent exo oxygen insertion.
`Thus, with norcamphor as substrate, P-450cam shows a drastic
`reduction in the fraction of 5-exo alcohol produced with inversion
`of configuration as compared to the substrate camphor. The
`deuterium data for the 6-alcohol reflect a similar preference for
`exo abstraction. The stereochemistry of hydrogen abstraction at
`It is apparent from the ex-
`the 3-position was not
`investigated.
`periment conducted in D20/buffer that the nearly complete loss
`of deuterium observed for the 5- and 6-alcohols is not due to an
`artefact of experimental workup. The background level of d\
`Consumed
`
`3757
`
`J. Am. Chem. Soc., Vol. 109, No. 12, 1987
`product obtained in D20 is most likely due to a minor degree of
`the 3-position. This is supported by the
`solvent exchange at
`observation that the 3-alcohol produced in D20 is less than 1%
`di-
`The importance of these stereochemical considerations becomes
`evident when the stoichiometry of norcamphor turnover
`as a
`function of substrate deuterium content
`is determined.
`Stoichiometry of Norcamphor Metabolism.
`It is qualitatively
`apparent from Figure 1 that there is a significant reduction in
`the amount of hydroxylated products obtained when hydrogen
`is substituted with deuterium on the substrate. This result ne-
`cessitated experiments in which the complete stoichiometry of this
`system was determined. Experiments were conducted in which
`the NADH and 02 consumptions were precisely quantitated along
`with formation of H2G2 and hydroxynorcamphor. Conditions were
`employed which insured that NADH was the limiting reagent.
`A similar reaction utilizing camphor as a substrate was conducted
`for comparison. A catalase assay was employed to convert in situ
`produced hydrogen peroxide to oxygen and water and was found
`to afford quantitative recovery of exogenously added H202 as
`demonstrated in Figure 3. The results from these measurements
`are summarized in Table III. When the sum totals of oxygenated
`products are compared to the absolute number of reducing
`equivalents consumed in the reaction, a striking unaccountability
`of electrons is observed. These are found to reside in excess water
`produced by four-electron reduction of dioxygen as described by
`Gorsky et al.2 In the presence of norcamphor, P-450cam displays
`a remarkable degree of oxidase activity with a correspondingly
`less efficient monooxygenase chemistry. A striking trend is ob-
`served when additional positions of norcamphor are deuteriated
`(Figure 4). With incremental addition of deuterium there is a
`corresponding decrease in the amount of hydroxylated product
`obtained. This cannot be accounted for by a significant increase
`in the amount of H2G2 produced, and, based on the 2:1 stoi-
`chiometry of excess NADH/excess 02,
`the decrease in hy-
`droxylation appears to coincide with an increase in oxidase-derived
`water production. These results demonstrate that, in addition to
`a deuterium isotope effect on the regioselectivity of hydroxylation,
`there exists an isotope-dependent switching from monooxygenase
`to oxidase activity at the active site of P-450cam. The near complete
`specificity for the exo face abstraction at the 5- and 6-positions
`in a maximal
`isotope effect on
`this metabolic
`should result
`switching when the 5,6-exo,exo-norcamphor-3,3,5,6-if4 is processed
`by the enzyme. Placement of deuterium at the endo face would
`Produced
`
`Figure 4. Deuterium isotope effects on the Stoichiometry of norcamphor metabolism.
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`3758
`
`J. Am. Chem. Soc., Vol. 109, No. 12, 1987
`
`Table III. Stoichiometry of Norcamphor Metabolism
`
`Atkins and Sligar
`
`substrate
`norcamphor
`
`I
`
`II
`
`III
`
`d-camphor
`
`catalase
`
`+
`
`+
`
`+
`
`+
`
`-
`
`-
`
`-
`
`-
`
`-
`
`h2o2,
`nmol
`88 ± 22
`
`84 ± 22
`
`ND
`
`74 ± 28
`
`20 ± 12
`
`produced
`ROH,
`nmol
`88 ± 12
`85 ± 6
`44 ± 2
`42 ± 6
`78 ± 9
`80 ± 5
`27 ± 4
`30 ± 5
`290 ± 2
`286 ± 9
`
`“extra HzO”,“
`nmol
`189
`
`[ , ,
`NADH -
`+ ROH1
`[H202 + ROH]
`02 -
`2.1
`
`210
`
`ND
`
`218
`
`0
`
`2.2
`
`2.1
`
`consumed
`NADH, nmol
`02, nmol
`577 ± 8
`365 ± 5
`567 ± 6
`321 ± 6
`583 ± 1
`338 ± 5
`570 ± 6
`296 ± 6
`562 ± 4
`ND4
`ND
`571 ± 5
`564 ± 15
`319 ± 4
`282 ± 10
`582 ± 4
`290 ± 8
`313 ± 8
`+
`318 ± 3
`280 ± 3
`“Determined from samples not containing catalase. 4ND = not determined.
`be expected to have a minimal effect, although a secondary isotope
`Scheme I. Proposed Branching of Monooxygenase vs. Oxidase
`effect might be detected.
`Activity0
`Discussion
`Several key points of departure between camphor and nor-
`camphor metabolism by the bacterial P-450cam system have been
`discovered. The multiplicity of metabolic options observed in the
`presence of norcamphor undoubtedly results from a lack of ab-
`solute complementarity between the enzyme active site and
`norcamphor. High-resolution crystal structures of both the
`camphor bound and the substrate free enzyme have been re-
`ported.11,12 Substrate specificity appears to be imposed by specific
`interactions including a hydrogen bond between Tyr 96 and the
`carbonyl moiety of camphor, van der Waals contact between Val
`295 and the substrate 8,9-gem-dimethyl group, and between the
`camphor 10-methyl group and the Leu 244-Val 247 hydrophobic
`cleft. Furthermore, there is no substantial conformational re-
`arrangement of the active site upon camphor binding, as indicated
`by a comparison between substrate free and substrate bound
`crystal structures. The major difference between the two structures
`seems to be in the amount of water present at the substrate binding
`pocket,12 which is consistent with the finding that differential spin
`states imposed by various camphor analogues are correlated to
`the degree of solvent exposure of the active site tyrosine and the
`on-rate for solvent binding to the heme iron.13 Obviously nor-
`camphor possesses the ability to hydrogen bond with Tyr 96, but
`any hydrophobic or steric interactions involving the methyl groups
`of camphor with the protein would be diminished. Our findings
`indicate that destruction of these interactions is sufficient to allow
`access of the 3- and 6-positions to the [FeO]3+ intermediate.
`It is also evident that the absence of these interactions has a
`significant effect on the stereochemical course of hydroxylation
`at the 5-position of norcamphor; the isosteric carbon of camphor
`experiences a significant degree of inversion of configuration
`whereas norcamphor does not. The 6-exo alcohol is derived with
`a similar stereochemical course.
`The amount of norcamphor-derived alcohol obtained from a
`fixed pool of NADH is nearly an order of magnitude less than
`the analogous product obtained from camphor. When one con-
`siders the uncoupling of pyridine nucleotide utilization and the
`small amount of product divided among three distinct species, it
`is not surprosing that norcamphor-derived products had previously
`been overlooked. These results demonstrate that in the presence
`of norcamphor, the majority of reduced oxygen intermediates go
`to produce H2G rather than H202 or organic alcohol. Although
`the kinetics of H2Q2, hydroxynorcamphor, and water production
`have not been examined in detail, preliminary data reported herein
`suggest that the rate of oxidase-derived water production is much
`faster in cytochrome P-450cam than it is with any of the hepatic
`cytochrome P-450’s. At saturating levels of norcamphor, puti-
`daredoxin, putidaredoxin reductase, and NADH, the rate of
`
`since the exact electronic configuration of this species is not estab-
`lished.
`NADH consumption by P-450cam is approximately 25-30 nmol
`of NADH/nmol of P-450/min (data not shown). Our data would
`suggest that the rate of oxidase-derived water production is about
`10 nmol of H20/nmol of P^SO^/min or about 33% of the total
`NADH consumption rate. Typical rates of NADPH consumption
`by various hepatic isozymes are slower than this value,14·15 as
`inferred from rates of product formation, so oxidase-derived water
`production is correspondingly quite slow compared to the P-dSO^
`It is especially interesting that the mononuclear metal
`system.
`center of cytochrome P-450cam, with no active-site acid/base
`groups,11·12 possesses the ability to reduce molecular oxygen to
`water at a relatively high rate.
`Deuteriation of the norcamphor skeleton has novel and profound
`effects on the stoichiometry of this system in addition to the
`It is unlikely that
`isotope-dependent alteration in regiospecificity.
`substrate deuteriation would have any appreciable effects on the
`kinetics or thermodynamics of substrate binding, electron transfer,
`or 02 binding, although each of these is expected to be different
`from the corresponding parameters in the presence of camphor.
`Indeed, others have observed that deuterium substitution does not
`lead to an increase in the level of H202 production or
`in the
`steady-state levels of oxy-P-450 intermediates.7 In view of these
`considerations, one would expect that the only isotopically sensitive
`step in the P-450 reaction cycle would be hydrogen abstraction
`by a compound I type high-valent iron-oxo species. There is
`precedent for two-electron reduction of this intermediate or hy-
`drogen atom abstraction followed by electron transfer from
`substrate radical in P-450 systems.16 This reflects the chemical
`competence of the iron-oxo species toward two-electron reduction.
`It has been proposed that a possible point for electron input to
`the level of this hy-
`result
`in an oxidase stoichiometry is at
`droxylating intermediate.2 Our data indicate that the species
`undergoing two-electron reduction is sensitive to deuterium sub-
`stitution of substrate and suggests that the [FeO]3+ reduction may
`be mediated by NADH and associated electron-transfer partners
`This may have some physiological significance.
`(Scheme I).
`Production of a highly electrophilic oxidant at the active site, in
`
`(11) Poulos, T. L.; Finzel, B. C.; Gunsalus, I. C.; Wagner, G. C.; Kraut,
`J. J. Biol. Chem. 1985, 260, 16122.
`(12) Poulos, T. L.; Finzel, B. C.; Howard, A. J. Biochemistry 1986, 25,
`53134.
`(13) Fisher,  . T.; Sligar, S. G. Biochemistry 1985, 24, 6696.
`
`(14) Koop, D. R.; Morgan, E. T.; Tarr, G. E.; Coon, M. J. J. Biol. Chem.
`1982, 257, 8472.
`(15) White, R. E.; McCarthy,  . B.; Egeberg, K. E.; Sligar, S. G. Arch.
`Biochem. Biophys. 1984, 228, 493.
`(16) Wand, M. D.; Thompson, J. A. J. Biol. Chem. 1986, 261, 14049.
`
`Apotex Ex. 1025
`
`

`

`Metabolic Switching in Cytochrome P-450cam
`the absence of an easily hydroxylatable substrate, would not be
`a desirable situation due to the potential for destructive oxidative
`reactions. The fact that an electron-transfer pathway remains
`open in the complete reconstituted system allows for rapid
`quenching of an active intermedíale in the absence of an acceptable
`substrate. This is consistent with the observation that multiple
`norcamphor turnovers do not result in any detectable levels of
`P-420 or of inactive enzyme. The decrease in product obtained
`with increasing amounts of deuteriation is not due to peroxide-
`[FeO]3+-dependent heme oxidation. This is further
`mediated or
`demonstrated by the fact that NADH is consumed to the same
`degree for each of the deuteriated substrates. Furthermore, a
`second addition of NADH, after completion of the reaction, results
`in continued NADH and 02 consumption at normal rates.
`Due to complexity of the multiple pathways available for the
`flux of reducing equivalents to form organic products, peroxide
`and water, it is difficult to calculate an intrinsic isotope effect for
`norcamphor hydroxylation. However, some kinetic information
`is available if a minimal kinetic scheme is analyzed (Scheme I).
`By comparing the oxygenase/oxidase ratio for undeuteriated and
`deuteriated substrate (III), one can obtain the lower limit for an
`intrinsic isotope effect which would be accurate if all three hy-
`droxynorcamphors were obtained from a single [FeO]3+-substrate
`complex with no kinetic barrier to reorientation.
`In this simple
`model, the relative amount of organic product and oxidase-derived
`water for these two substrates is given by [AtH/Atw]/[A:D//cw], where
`kH and kD represent the rate constants for hydrogen or deuterium
`abstraction and kw represents the rate constant for reduction of
`the intermediate to water.
`This second rate, kw, whould be
`insensitive to isotopic substitution, and if insensitive to norcamphor
`orientation, would be equivalent for the two substrates. The ratio
`of oxygenase/oxidase activity, as measured by the ratio of total
`formation, should offer
`organic alcohol production to total water
`a measure of the intrinsic isotope effect kH/kO. This value is 3.78
`Intrinsic isotope effects for P-
`for the data presented herein.
`450-mediated hydroxylations have been estimated by several
`methods with values ranging from 7.5 to 19.6·8,17 These large
`isotope effects are expected for a radical mechanism involving
`hydrogen abstraction. The relatively small value obtained by this
`simplified kinetic scheme would suggest that a high degree of
`masking is operational and that reorientation of norcamphor occurs
`faster than, reduction of the iron-oxo
`on the same time scale as, or
`intermediate.
`Several observations support the conclusion that H202 is not
`an intermediate in the four-electron reduction of 02 in the P-450cam
`system. Exogenously added H202 had no effect on the rate or
`amount of NADH and 02 consumption with any of the nor-
`camphor analogues utilized. Furthermore, it is unlikely that the
`hydroxylation was actually mediated by H202 since exogenously
`added hydrogen peroxide or catalase had no effect on the amount
`of hydroxynorcamphor obtained.
`It is possible that an enzyme-
`bound H202 is generated followed by rapid peroxidase-type
`formation of a higher valent Compound I type intermediate. Such
`If this type
`a fate for H202 would not be detected by our assay.
`of mechanism were operative then these results still suggest that
`this intermediate was a substrate for further two-electron reduction
`to form water.
`It is worth noting that the experiments reported herein utilized
`It is possible that the enantiomers of this
`racemic norcamphors.
`substrate are processed with different kinetic parameters and that
`the oxygenase/oxidase ratio may be enantiomer dependent.
`Although these various alternatives are remotely feasible, they
`do not affect the proposal that an oxidase activity exists in the
`P-450cam isozyme and that this activity involves reduction of
`[FeO]3+ via NADH and the associated electron-transport chain.
`Finally, several questions are raised by the observation that
`norcamphor turnover elicits a high degree of oxidase activity. The
`structure of norcamphor would be expected to result in an active
`site complex with two features significantly different from that
`
`(17) Groves, J. T.; McCluskey, G. A.; White, R. E.; Coon, M. J. Biochem.
`Biophys. Res. Commun. 1978, 81. 154.
`
`J. Am. Chem. Soc., Vol. 109, No. 12, 1987
`
`3759
`
`formed with camphor. Firstly, on the basis of the percent ferric
`high-spin complex (45%) obtained with saturating norcamphor,
`it would be expected that a significant amount of water remained
`at the active site in the presence of this substrate.12,13 Hydration
`of the [FeO]3+ species may allow for, and even be required for,
`reduction to H20, since protons would be required to completely
`balance the oxidase stoichiometry. Thus, the relative rate for the
`reduction of [FeO]3+ may be a function of the degree of hydration
`of this intermediate. Secondly, one can imagine that there is an
`increase in the amount of substrate