7928
J . Am. Chem. SOC.1984, 106, 1928-1935
Mechanistic Studies on Non-Heme Iron Monooxygenase Catalysis: Epoxidation, Aldehyde Formation, and Demethylation by the w-Hydroxylation System of Pseudomonas oleovorans Andreas G. Katopodis, Kandatege Wimalasena, Joseph Lee, and Sheldon W. May* Contribution from the School of Chemistry, Georgia Institute of Technology, Atlanta, Georgia 30332. Received April 9, 1984
Abstract: In previous work we have established that the "w-hydroxylation" system of P.oleouorans readily converts terminal olefins to the corresponding 1,2-oxides and does so stereoselectively. We also demonstrated loss of olefin configuration during enzymatic epoxidation, a result inconsistent with a concerted epoxidation mechanism ( J . Am. Chem. SOC.1977,99, 2017-2024). Since loss of olefin configuration is unprecedented for monooxygenase-catalyzedepoxidations, these studies have been confirmed with isolated enzymes and further extended in order to probe the mechanism of non-heme iron monooxygenase catalysis. Enzymatic epoxidation of both cis- and trans-1-deuterio- 1-octene proceeds with about 70% inversion of the olefinic configuration, with corresponding results being obtained for the two olefins. As we reported in a preliminary communication (Bio/ Technology 1983, I , 677-686), the w-hydroxylation system also produces aldehydes from olefins. Aldehyde formation exhibits the reaction characteristics expected for the usual oxygenase pathway. Deuterium migration from C-1 to C-2 occurs in formation of aldehyde from olefin, although loss of deuterium also occurs. The w-hydroxylationsystem was found to efficiently catalyze 0-demethylation of heptyl methyl ether, the first demonstration of such activity for a non-heme iron monooxygenase of this type. Taken together, the results provide support for a two-step mechanism involving enzyme-generated species with cationic and/or radical character, which accounts for the stereoselectivity,configurational loss, substratespecificity, formation of aldehydes with deuterium migration, and demethylation activity exhibited by this enzyme system.
The mechanism by which oxygenases catalyze the insertion of molecular oxygen into organic molecules has been the subject of intense scrutiny in recent years, due to the importance of these enzymes in detoxification, oncogenesis, biosynthesis, and metabolism in general. However, while the P-450 and the flavin-containing monooxygenases have been intensively studied at the molecular level, the state of our understanding of the molecular basis of non-heme iron monooxygenasecatalysis is, by comparison, poor indeed. Non-heme iron monooxygenases comprise a large class of enzymes which, in addition to the P. oleouorans system discussed here, includes the phenylalanine, tyrosine, and tryptophan monoxygenases as well as squalene epoxidase.1,2 The non-heme iron monooxygenase system from P.oleouorans which catalyzes terminal methyl group hydroxylation of alkanes and fatty acids was first shown by Coon and co-workers to consist of three protein components: rubredoxin, a flavoprotein reductase, and non-heme iron m o n ~ x y g e n a s e . ~ -In ~ ~previous work from
(1) Comprehensive reviews on oxygenases can be found in: (a) "The Enzymes", 3rd ed.;Boyer, P. D.,Ed.; Academic Press: New York, 1975; vol. 12. (b) 'Molecular Mechanisms of Oxygen Activation"; Hayaishi, O., Ed.; Academic Press: New York, 1974. Also, for a review of biological hydroxylation reactions, see: (c) Gunsalus, I. C.; Pederson, T. C.; Sligar, S.G. Annu. Reu. Biochem. 1975,44,377-407. (d) A review on P-450oxygenation in: White, R. E.; Coon, J. J. Annu. Rev. Biochem. 1980, 49, 315-356. (2) For a recent review on enzymatic epoxidation reactions, see: May, S. W. Enzyme Microb. Technoi. 1979, I , 15-22. (3) Peterson, J. A.; Basu, D.; Coon, M. J. J. Bioi. Chem. 1966, 241, 5162-5164. (4) Peterson, J. A.; Kusunose, M.; Kusunose, E.; Coon, M. J. J . Bioi. Chem. 1967, 242,4334-4340. (5) Peterson, J . A.; Coon, M. J. J. Bioi. Chem. 1968, 243, 329-334. (6) McKenna, E. J.; Coon, J. J. J . Biol. Chem. 1971, 245, 3882-3889. (7) Lode, E. T.; Coon, J. J. J. Bioi. Chem. 1971, 246, 791-802. (8) Ueda, T.; M e , E. T.; Coon, J. J. J. Bioi. Chem. 1972,247,2109-21 16. (9) Benson, A,; Tomoda, K.; Chang, J.; Matsueda, G.; Lode, E. T.; Coon, M. J.; Yasunogu, K. T. Biochem. Biophys. Res. Commun. 1971,42,640-646. (10) Boyer, R. R.; Lode, E. T.; Coon, M. J. Biochem. Biophys. Res. Commun. 1971.44, 925-930. (11) Ueda, T.; Coon, M. J. J. Bioi. Chem. 1972, 247, 5010-5016. (12) Ruettinger, R. T.; Olson, S. T.; Boyer, R. F.; Coon, M. J. Biochem. Biophys. Res. Commun. 1974, 57, 1011-1017. (13) Ruettinger, R. T.; Griffith, G . R.; Coon, M. J. Arch. Biochem. Biophys. 1977, 183, 528-537.
this laboratory, we demonstrated that this enzyme system readily converts terminal olefins to the corresponding 1,2-epoxides, and does so stereoselectively,producing the R-(+) epoxides.14-'* We have also examined the specificity of this r e a c t i ~ n 'and ~ * ~have ~ carried out immobilization, metal substitution, and chemical modification experiments with r ~ b r e d o x i n . ~ l -However, ~~ the instability of the monooxygenase hampered detailed mechanistically oriented experiments with the isolated system, a common problem with the non-heme iron monooxygenases. We therefore turned to the use of resting whole cells and demonstrated through the use of the substrate trans,trans- 1,8-dideuterio-1,7-0ctadiene that enzymatic epoxidation does not proceed with retention of the original olefin geometry.24 Taken together, the combined stereoselectivity of oxygen attack at carbon 2 and lack of configurational retention at carbon 1 are inconsistent with a concerted "oxenoid" mechanism, and we suggested possible two-step mechanisms involving cationic and/or radical intermediates. The goals of the present study were to confirm and extent these mechanistic studies using purified reductase and rubredoxin and a partially purified preparation of the monooxygenase. We wished, first of all, to demonstrate loss of olefin configuration with both cis- and trans-deuterated substrates and to establish whether complementary ratios of cis- and tram-epoxides would be obtained from these two substrates. This was an important goal, since the (14) May, S. W.; Abbott, B. J. Biochem. Biophys. Res. Commun. 1972, 48, 1234-1234. (15) May, S. W.; Abbott, B. J. J. Bioi. Chem. 1973, 248, 1725-1730. (16) May, S. W.; Schwartz, R. D. J. Am. Chem. SOC. 1974, 96, An7 1 -407 ._--.7 . " _ I
(17) May, S. W.; Steltenkamp, M. S.; Schwartz, R. D.; McCoy, C. J. J . Am. Chem. SOC.1976, 98, 7856-7858. (18) May, S. W. Caral. Org. Synth. [Con/.] 4rh, 1976 1977, 101-111. (19) May. S.W.; Schwartz. R. D.;Abbott, B. J.; Zaborsky, 0. R. Biochem. Biophys. Acra 1975, 403, 245-255. (20) May, S. W.; Steltenkamp, M. S.; Boram, K. R.; Katopodis, A. G.; Thowsen, J. R. J. Chem. Soc., Chem. Commun. 1979, 845-846. (21) May, S. W.; Kuo, J. Y. J. Bioi. Chem. 1977, 252, 2390-2395. (22) May, S. W.; Kuo, J. Y. Biochemistry 1978, 17, 3333-3338. (23) May, S. W.; Lee, L. G.; Katopodis, A. G.; Kuo, J. Y.; Wimalasena, K.; Thowsen, J. R. Biochemistry 1984. (24) May, S. W.; Gordon, S. L.; Steltenkamp, M. S. J. Am. Chem. Soc. 1977, 99, 2017-2024.
0002-7863/84/ 1506-7928$01.50/0 0 1984 American Chemical Society
Mechanistic Studies on Non- Heme Iron Monooxygenase Catalysis loss of olefin configuration during epoxidation is, to our knowledge, unprecedented for any other monooxygenase system described in the literature. Second, as we have recently reported,25preliminary studies with the purified P.oleouorans monooxygenase (POM) indicated formation of aldehydes, in addition to epoxides, from terminal olefins. Since aldehyde formation could arise as a consequence of group migration in an enzymatically produced species with cationic character, we wished to examine the aldehyde formed from 1,I-dideuterio-1-octene in order to obtain direct evidence for intramolecular rearrangement. Finally, in order to obtain further evidence regarding the locus of initial oxygen attack, the possibility that POM could catalyze demethylation reactions with appropriate substrates was investigated. The results reported herein have allowed us to propose a mechanism of oxygenation by this non-heme iron monooxygenase, which is consistent with all stereochemical and mechanistic results so far reported.
Experimental Section Hydrocarbons, solvents, organic reagents, and standards were purchased from various sources and were of the highest purity available. Authentic epoxide standards were synthesized by using m-chloroperbenzoic acid and the appropriate olefins, as described previ0us1y.l~ P. oleouorans cultures were grown on n-octane under conditions described elsewhere.24 P. oleouorans rubredoxin was purified by using a procedure described by us earlier.21a22Spinach ferredoxin-NADP' reductase, glucose-6-phosphate dehydrogenase, and all other biochemicals were purchased from Sigma. Reaction conditions for standard product-formation assays as well as procedures for extraction and quantitation of products were as described, except in some cases where, as indicated, the extraction solvent was changed to facilitate extraction and concentration of the various products. Synthesis of trans-1-Deuterio-1-octene.The procedure developed by Brown and Gupta was used.26 The crude product was distilled, bp 121 OC (760 mm), and further purified by preparative G C (20-ft carbowax 20 M, 90 "C). The final product consisted of a single peak with retention identical with the fully protonated 1-octene standard. The N M R was consistent with the expected structure and showed 94% deuterium in the trans position, and the cis isomer was not detectable. Synthesis of I-Deuterio-I-octyne. A solution of 11 g (100 mmol) of freshly distilled 1-octyne in 100 mL of anhydrous ether was cooled to 0 OC in an ice-salt bath and 1.1 equiv of freshly titrated methyllithium in ether (purchased from Aldrich) was added dropwise under argon. The reaction mixture was stirred 3 h at 0 OC and allowed to gradually warm to room temperature. D 2 0 , 3.0 g (100% D), was then added, stirred for h at room temperature, and neutralized with 10% acetic acid. The reaction mixture was poured into ice-cold water and extracted thrice with 25-mL aliquotes of ether, and the combined extracts were washed with saturated NaHCO, and saturated brine, dried over anhydrous MgSO,, and distilled to give 9 g of I-deuterio-I-octyne (bp 125 OC (760 mm)). N M R analysis indicated that the product was 1-deuterio-1-octyne with only 80% deuterium incorporated. The material was again reacted with methyllithium and D 2 0 , and after distillation 6 g of material was obtained with 99% D incorporation (NMR). Gas chromatographic analysis indicated that the product consisted of one peak with retention time identical with a 1-octyne standard. Synthesis of 1,l-Dideuterio-I-octene. 1-Deuterio-1-octyne, 5 g, was reacted with catechol borane and then deuterated acetic acid as indicated for I-deuterio-1-octene. The olefin was isolated by preparative gas chromatography, and N M R analysis showed 99% deuterium in the cis position and 86% deuterium in the trans position. Synthesis of cis-I-Deuterio-I-octene.1-Deuterio-1-octyne, 5 g, was reduced as above, using protioacetic acid in the final step. The resulting product exhibited one peak coeluting with 1-octene on the GC. Mass spectral analysis was consistent with 1-deuterio-1-octene, and N M R analysis indicated that 99% deuterium was present in the cis position and no trans was detectable. Synthesis of trans -1,2-Epoxy-l-deuteriooctane, cis -1,2-Epoxy-ldeuteriooctane, and 1,2-Epoxy-l,l-dideuteriooctane. A solution of each olefin (0.06 mL, 0.38 mmol) and m-chloroperbenzoic acid (100 mg, 0.58 mmol) in 14 mL of CH2CI2was stirred at room temperature overnight, after which it was diluted with 25 mL of CH2C1,, washed with sodium bisulfite and NaHCO,, and then dried over anhydrous MgSO,. The concentrated product was purified by preparative gas chromatography to obtain high-purity epoxides.
J . Am. Chem. SOC.,Vol. 106, No. 25, 1984 7929 POM Partial Purification. The procedure described here is a modification of processes published elsewhereI3 and of our previous proced u r e ~ and ~ ' affords a form of enzyme stable and active enough to produce milligram quantities of products. Two hundred grams of P.oleouorans cells were lysed for 2 h in 1.5 L of 20 mM Tris CI, pH 7.4 (all operations were performed at 4 OC, and the pH was adjusted at every step). The solution was centrifuged and the membrane pellet was lyseu again for another 2 h. After centrifugation the membrane pellet was resuspended in 600 mL of the same buffer and sonicated in 1-min blasts for a total of 5 rnin with a Branson Sonifier. The solution was diluted with 600 mL of cold buffer and centrifuged (20000 g, 1 h), and the supernatant was subjected to ammonium sulfate precipitation. The 30-35% pellet was resuspended in 50 mL of 20 mM Tris CI, 20% glycerol, 0.1% deoxycholate, pH 7.4 buffer and centrifuged again (50000 g, 2 h), and the supernatant was assayed and stored at -70 "C. The best preparations had a specific activity of 465 nmol per mg of protein of total (both epoxide and aldehyde) product in the standard G C assay with 1,7-octadiene as a s u b ~ t r a t e . l ~ ~ ~ ~ Assays. In a total volume of 1 mL of buffer (100 mM Tris CI, pH 7.4) were added 1 mg of partially purified P. oleouorans monooxygenase, 80 wg of P. oleouorans rubredoxin, 14 wg spinach ferredoxin reductase, 0.5 mg NADPH and 20 fiL of substrate solution (100 pL/l.OO mL of acetone). The mixture was shaken at 25 OC for 10 min. It was then extracted with 200 pL of hexane by using 2-octanol as the internal standard, and the hexane layer was analyzed by GC. Whenever NADPH recycling was needed 2 units of glucose-6-phosphate dehydrogenase and 2 mg of glucose 6-phosphate were included in the incubation mixture. The crude system was obtained by suspending 100 g of cells in 300 mL of buffer. The solution was sonicated for 7 min in 1-min blasts. It was then centrifuged (20000 g, 1 h), and the cloudy supernatant was again centrifuged (35 000 g, 2 h) to ,,btain a clear yellow-pink solution. This clarified cell extract was very stable upon freezing and was assayed by adding 0.20 mL of cell extract to 0.80 mL of buffer and 20 fiL of substrate solution. The assay mixture was then incubated and extracted as for the partially purified system. Whenever detection of carboxylic acids was required, after the incubation time was completed the assay mixture was basified to pH > 12 and washed with an equal volume of pentane, and the mixture was then acidified to pH
