Studies of the Mechanism of Oxygen Activation and Transfer

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Studies of the Mechanism of Oxygen Activation and Transfer Catalyzed by Cytochrome P 450 JOHN T. GROVES, S. KRISHNAN, GUILLERMINA E. AVARIA, and THOMAS E. NEMO Department of Chemistry, University of Michigan, Ann Arbor, MI 48109

The oxidation of cyclohexene by reconstituted and peroxide-dependent cytochrome P-450 systems has been examined. The ratio of cyclohexene oxide to cyclohexenol has been shown to vary with changes in the oxygen donor used. The hydrogen-isotope effect for allylic hydroxylation has been found to be 5 ± 0.5 and independent of oxygen donor. The changes in product ratio are attributed to the presence of the oxygen donor at the active site during oxygen transfer. The invariance of the isotope effect supports a similar transition state for the NADPH/O -dependent and peroxide-dependent paths. Treatment of tetra-o-tolylporphinatoiron(III) chloride with iodosylbenzene produces a transient intermediate that transfers oxygen to substrates and degrades the porphyrin in the absence of substrate. LM2

2

T

he role of cytochrome P 450 , the heme-containing, mixedfunction oxidase of liver microsomes, in catalyzing the oxidative transformations of lipid metabolism and the oxidative detoxification of drugs and other xenobiotic substances has been studied extensively (I, 2, 3). It is understood now that two successive, one-electron reductions of the heme center of cytochrome P 450 bind and reduce molecular oxygen to the formal oxidation state of hydrogen peroxide. A number of lines of evidence suggest that the active oxygen species in the P-450 cycle is a ferryl-ion intermediate equivalent to F e 0 formed by the heterolysis of the O - O bond of hydrogen peroxide {4, 5, 6). The important observation that cytochrome P 450 will catalyze the LM

3 +

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Dolphin et al.; Biomimetic Chemistry Advances in Chemistry; American Chemical Society: Washington, DC, 1980.

278

BIOMIMETIC CHEMISTRY

anaerobic oxidation of hydrocarbons in the presence of hydroperoxides (5, 7, 8) has offered persuasive evidence in favor of an overall reaction cycle such as that outlined in Scheme 1.


Scheme 1. Oxygen activation and transfer by cytochrome P 450

Speculation on the mechanism of oxygen insertion into unactivated C - H bonds catalyzed by cytochrome P 450 has centered on the apparent retention of configuration at the carbon and the low isotope effects generally observed for these processes (9, 10). We and others recently have demonstrated that with appropriately designed molecular probes very large intramolecular discrimination isotope effects can be observed (J J, 12). The oxidation of norbornane with cytochrome P 450 gave only exo- and endo-2-norborneol with an isotope effect of 11.5. Close examination of the mass spectra of the products revealed that, though the hydroxylation proceeded with predominant net retention of configuration at the carbon, a substantial fraction (up to 25%) of the oxidation had occurred with loss of stereochemistry at the oxidized carbon. The magnitude of this isotope effect and the partial loss of stereochemistry are similar to results observed for the oxidation of alkanes by other transition-metal-oxo reagents such as chromate and permanganate (13, 14). The free-radical mechanism proposed by Wiberg (15) for these chemical oxidations is consistent with the observations noted above for cytochrome P 450 and is depicted in Scheme 2 (16, 17). Although the peroxide shunt path to a ferryl intermediate is an attractive hypothesis, there is very little data to support the idea that the species obtained by this path are the same as, or similar to, that LM2


15.

GROVES E T AL.

279

Oxygen Activation 6- Transfer

produced by the fully reconstituted system involving N A D P H , P-450 reductase, and 0 . We report here a comparison of the aerobic and peroxide-dependent oxidations of cyclohexene by cytochrome P 450 2 (18,19). Product selectivities have been shown to be a function of the structure of the oxygen donor, whereas the magnitude of the hydrogen isotope effect for hydroxylation is not, 2

LM


Scheme 2. : 6 :

P-450-Fe

H-r

P-450-Fe

—

I V

/

0-H I iv

7

P-450-Fe

HC> P-450-Fe

1 1 1

+

Oxidation of Cyclohexene Catalyzed by Cytochrome P 450 The oxidation of cyclohexene by a fully reconstituted cytochrome P-450 system (5) gave only cyclohexene oxide (1) and cyclohexenol (2) in a ratio of 0.92: 1. Results for the peroxide-dependent oxidation of cyclohexene in the presence of cytochrome P 450 are presented in Table I. Inspection of the data suggests that subtle differences exist between the oxidants generated from these four oxidants and lead to the observed sixfold change in the ratio of 1 and 2 in the product mixture. Also apparent is the fact that no obvious correlation exists between the ratio of the products and the nature or effectiveness of the oxidant. LM

Table I.

Oxidation of Cyclohexene Catalyzed by Cytochrome P 450

Oxygen Donor

Cyclohexene Oxide (1) (nmol)

Cyclohexenol (2) (nmol)

1 2

Relative Efficiency

Cumene hydroperoxide f-Butyl hydroperoxide Iodobenzene diacetate Iodosylbenzene

122 3.2 7.2 25.8

138 12.1 39.1 26.1

0.88 0.26 0.18 0.99

17.3 1.0 3.1 3.5

This variability in the product ratios with the structure of the oxygen donor requires that the oxidant produced by these reagents be different in some functional way. The simplest explanation is that the


280


alcohol or iodobenzene fragment (X) is still at the active site when oxygen transfer takes place. Two extremes of the mechanistic spectrum that are consistent with these observations are: (a) the interaction of the substrate with the peroxidic oxygen occurring before the O - X bond is broken (3), and (b) production of a ternary cage structure (4) withX and the substrate (S) both at the active site (Scheme 3).


Scheme 3.

\

S

:X

/

4 For Path (a), the structure of X would be expected to exert a profound effect on the nature of the oxidizing intermediate. In Path (b), the effect of X may be only to affect the population of various substrate-active site complexes. Hydrogen-Isotope Effect for Hydroxylation of Cyclohexene We have examined the hydrogen-isotope effect for cyclohexenol formation as a probe of the nature of C - H bond cleavage in the NADPH-dependent and cumene-hydroperoxide-dependent oxidation of cyclohexene. Results for the oxidation of cyclohexene, 1,3,3-trideuterocyclohexene (5) and 3,3,6,6-tetradeuterocyclohexene (6) are given in Table II. It is apparent from the large increase in the relative amount of cyclohexene oxide upon deuteration of the allylic hydrogens that the hydroxylation path has a significant hydrogen-isotope effect (U, 12).


15.

GROVES ET AL. Table II.

281

Oxygen Activation ir Transfer

Effect of Deuteration on Cyclohexene Oxidation by Cytochrome P 450 LM2

Oxygen Donor x

Substrate , ,

NADPH/O2 I

A

n

p

u

/

4

5 6 cyclohexene 5

3

15.3

g

4

39.0 14.2 44.4 38.4 36.3 1.10 1.8

cyclohexene 6

g

1 2 0.89

a

Q gj

Q

25!5 2.97° 48.2 25.9 8.1 3.68 1.46

a

6

t-Butyl hydroperoxide

Cyclohexenol (2)

12.0°

cyclohexene

n

Cumene hydroperoxide Downloaded by STONY BROOK UNIV SUNY on October 6, 2017 | http://pubs.acs.org Publication Date: December 10, 1980 | doi: 10.1021/ba-1980-0191.ch015

Cyclohexene Oxide (1)

L5 4.7 0.92 1.48 4.51 0.29 1.23

Absolute product yields were found to be related to the age and batch of NADPHcytochrome P-450 reductase. a

The magnitude of the isotope effect can be derived from the appropriate competitive rate expression for any two of the product ratios. The results of these calculations are given in Table III. An independent determination of the isotope effect could be derived from the mass spectrum of the 3-trimethylsiloxycyclohexene-d„ (7) obtained from the oxidation of 5. Authentic 3-trimethylsiloxycyclohexene-d (8) gave prominent ions corresponding to the molecular ion (m/e = 170) and loss of ethylene by retro Diels-Alder fragmentation (m/e = 142). 0

Table III.

Isotope Effects for Cyclohexene Hydroxylation by Cytochrome P 450 LM2

Isotope Effect (k /k ) H

Rate Expression

a

"

k

/ k D =

»°

k

/k

D

2

%u *

= 2[1,

k /k = H

2[i/2u -mu 2]

NADPHI0

2

4

1

4 9

4.9

P-450-CHP System Cumene Hydroperoxide

D

t-Butyl Hydroperoxide

-

4

8

5 1

4.9

4.25

For simplicity, in the derivation of isotope effect expression, possible secondary deuterium isotope effects have been ignored. a


282


The mass spectra of 7, obtained from the NADPH/O /P-450 oxidation of 5, and 8 are compared in Table IV. Three isotopic species are expected for the allylic hydroxylation of 5. Oxidation with removal of hydrogen will produce 7-d and 7a-d . Extensive controls have demonstrated that 7-d and 7a-d are indistinguishable by mass spectrometry because of rapid 1,3-migration of the siloxy group upon electron impact Oxidation with deuterium removal leads to the production of 7-d (Scheme 4). By this analysis, the isotope effect is simply the ratio of 5-d (and 5a-d ) to 5-d in the oxidized sample. Deconvolution of the parent region (with appropriate correction for carbon and silicon 2

3

3

3

3

2

3


3

Table IV.

2

Mass Spectra of 3-Trimethylsiloxycyclohexenes Derived from the P-450 -Catalyzed Oxidation of 5 LM2

7-d from 5

] m/e jlntensity

173 100

172 46.3

1 m/e , , \lntensity nexenol J

170 100

169 37.3

n

, J

v

k /k H

D

171 19.4

145 31.8

144 18.8

143 26.2

143 6.5

142 45.2

141 4.0

k /k

= 6.0

H

D

= 4.5

Scheme 4. OSiMe,

OSiMe,

(1)

m/e=172

m/e=14A

(2)

m/e=173

OSiMe,

m/e=145

7-d,


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Oxygen Activation &• Transfer

283


isotopes) gave an isotope effect of 6.0 for the allylic hydroxylation of cyclohexene by the reconstituted system. Similar treatment of the retro Diels-Alder fragment gave a value of 4.5.

5

6

Thus, the isotope effect for the allylic oxidation of cyclohexene by cytochrome P 450 is about 5 and is the same for the reconstituted, NADPH-dependent and the peroxide-dependent paths. This similarity suggests that although product ratios may change from one oxygen donor to another, the mechanism of oxygen transfer may be invariant. Efforts to develop a clearer understanding of the relationships between the 0 -dependent and peroxide-dependent pathways for oxygen transfer catalyzed by cytochrome P 450 are currently underway. 2

Oxygen Transfer Catalyzed by Synthetic Metalloporphyrins The observation that cytochrome P 450 can be driven by hydroperoxides and related oxygen donors suggests that metalloporphyrins can be made to function as oxygen-transfer catalysts in simple model systems. In early attempts to produce an iron-oxo species (20) from typical porphyrins like chloro-a,/3,y,8-tetraphenylporphinatoiron(III) [Fe(III)TPP-Cl] andchloroferriprotoporphyrin(IX)[Fe(III)PPIX-Cl], we examined the reaction of f-butyl hydroperoxide and peroxyacids with alkanes and olefins in the presence of these catalysts. With peroxyacids, decomposition of the porphyrin ring was observed, while with the t-butyl hydroperoxides, product distributions were indistinguishable from free-radical chain reactions initiated photochemically in the absence of any metals. Therefore, it appears that the redox properties of the metalloporphyrin are required only for the initiation step in these free-radical autoxidations and that the porphyrin is not a stoichiometrically significant catalyst (21, 22, 23). The failure of these simple approaches to a reaction iron-porphine oxide or its equivalent could indicate that the ligation state of iron in the protein, presumably including an axial thiolate, is crucial to the oxygen-transfer properties of P 450. Support



284


for this idea can be derived from the observation that cytochrome P 420, a partially denatured form of P 450, is ineffective as a catalyst for the oxidation of cyclohexene either in the fully reconstituted system or in the peroxide-dependent route. Another interpretation, however, is that the heme unit in P 450 effectively suppresses the freeradical chain reactions that dominate the chemistry in solution. We have demonstrated recently that epoxidation and hydroxylation can be achieved with simple iron-porphine catalysts with iodosylbenzene as the oxidant (24). Cyclohexene can be oxidized with iodosylbenzene in the presence of catalytic amounts of Fe(III)TPP-Cl to give cyclohexene oxide and cyclohexenol in 55% and 15% yields, respectively. Likewise, cyclohexane is converted to cyclohexanol under these conditions. Significantly, the alcohols were not oxidized rapidly to ketones under these conditions, a selectivity shared with the enzymic hydroxylations. The distribution of products observed here, particularly the preponderance of epoxide and the lack of ketones, is distinctly different from that observed in an autoxidation reaction or in typical reactions of reagents such as chromates or permanganates (15).

The oxidation of dioctyl Fe(III)PPIX-Cl with iodosylbenzene (9) showed that the octyl sidechains had been hydroxylated and that 60% of the hydroxylation had occurred at C(4) and C(5) in the middle of the chain. Molecular models indicate that these two carbon centers have the most favorable access to the center of the porphyrin ring, supporting the idea that the mechanism of this hydroxylation is an intramolecular oxygen rebound (25, 26) from iodine to iron and into the C - H bond (Scheme 5).


15.

GROVES ET AL.

Oxygen Activation b- Transfer

285


Scheme 5.

Generation of an "Active Oxygen" Metallo-Porphyrin Intermediate Treatment of TPPFe(III)Cl or raeso-tetra-o-tolylporphinatoiron(III) chloride [TTPFe(III)Cl (10)] with iodosylbenzene caused rapid oxidation of the porphyrin and loss of catalytic activity for hydrocarbon oxidation. Figure 1 shows changes in the visible absorption spectrum upon treatment of 10 with iodosylbenzene. These data indicate that shortly after the addition of iodosylbenzene (Scan b, Figure 1) a new porphyrin species (11) is formed, which then rapidly decays to oxidized porphyrin products. The kinetics of this decay process are approximately first order (Figure 2). A mechanism for oxygen transfer catalyzed by 10 consistent with these data is given in Scheme 6. It is clear that iodosylbenzene reacts with simple iron-porphine complexes to form a reactive intermediate (11). This intermediate is capable of transferring oxygen to substrates such as cyclohexene and cyclohexane. In the absence of substrate, oxygen apparently is transferred to the porphyrin ring. It is not possible to assign unambiguously the structure of the reactive intermediate 11 at this time. Likely formulations are a 1:1 complex between the iron(III) porphyrin and iodosylbenzene or an iron-oxo intermediate equivalent to iron(V). The nature of this intermediate and its relevance to biological-oxygen activation and transfer are under continued study in our laboratories.



286


Figure 1. Visible spectrum of TTPFe(III)Cl and iodosylbenzene in methylene chloride: Scan a, time = 0; Scan b, time = 13 sec; Scan c, time = 25 sec; Scan d, time = 35 sec Methods and Materials Deuterated Cyclohexenes. 1,3,3-Trideuterocyclohexene (5) was synthesized from cyclohexanone according to the method of Fahey and Monahan (27) except that diazabicyclononane (DBN) was used instead of potassium f-butoxide in the final step. 3,3,6,6-Tetradeuterocyclohexene (6) was obtained from Merck, Sharp, and Dohme (>98

Studies of the Mechanism of Oxygen Activation and Transfer

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