Radical intermediates in monooxygenase reactions of Rieske dioxygenases

Article abstract of DOI:10.1021/ja068188v

Rieske dioxygenases catalyze the cis-dihydroxylation of a wide range of aromatic compounds to initiate their biodegradation. The archetypal Rieske dioxygenase naphthalene 1,2-dioxygenase (NDOS) catalyzes dioxygenation of naphthalene to form (+)-cis-(1R,2S

Full text of DOI:10.1021/ja068188v

Published on Web 03/07/2007
Radical Intermediates in Monooxygenase Reactions of Rieske Dioxygenases
Sarmistha Chakrabarty,§ Rachel N. Austin,‡ Dayi Deng,¶ John T. Groves,¶ and John D. Lipscomb*,§
Department of Biochemistry, Molecular Biology, and Biophysics, UniVersity of Minnesota, Minneapolis,
Minnesota 55455, Department of Chemistry, Bates College, Lewiston, Maine 04240, and Department of Chemistry,
Princeton UniVersity, Princeton, New Jersey 08544
Received November 15, 2006; E-mail: lipsc001@umn.edu
Table 1. Product Distribution from Probe Oxidation by NDOS
Rieske cis-diol forming dioxygenases are among the most
versatile of nature’s oxidizing catalysts and are often employed to
initiate the biodegradation of recalcitrant environmental pollutants.1
The natural substrates for these enzymes are unactivated aromatic
compounds. These are converted to nonaromatic cis-dihydrodiols
with the incorporation of both oxygen atoms from O2 in a reaction
coupled to the oxidation of NADH.
Naphthalene 1,2-dioxygenase is a Rieske dioxygenase that
catalyzes the oxidation of its name substrate to form (+)-cis-
(1R,2S)-dihydroxy-1,2-dihydronaphthalene.1b The enzyme system
(NDOS) consists of an FAD and Fe2S2 cluster-containing reductase
(NDR), a ferredoxin (NDF), and an R3â3 oxygenase (NDO)
containing a Fe2S2 Rieske cluster and a mononuclear iron center
in each R subunit. NDOS exhibits a broad substrate range for
aromatic cis-dihydroxylation.1c Moreover, it catalyzes monooxy-
genase reactions with a diversity rivaling cytochrome P450 (P450)
and methane monooxygenase (MMO).1b,c,2
The mechanism for oxygen insertion by Rieske dioxygenases is
unknown for both the di- and monooxygenation reactions. Con-
certed mechanisms as well as those enlisting cation or radical
intermediates have been proposed.1d,3 Here, we examine the
mechanism of the monooxygenase reaction of NDOS using the
diagnostic probes norcarane (1) and bicyclohexane (6). These give
rearranged or ring-expanded products when radical or cation
intermediates, respectively, are formed.4 For radical intermediates,
the ratio of unrearranged to rearranged products allows estimation
of the radical lifetime. Failure to observe reorganized products
suggests a concerted mechanism or a short-lived intermediate. It is
shown here for the first time that Rieske dioxygenases catalyze
monooxygenase reactions by formation of a substrate radical
intermediate. These results may hold implications for the mechanism
of dioxygenation by this family.
The oxidation of 1 and 6 yielded the endo- and exo-2-alcohols
2a, 2b, 7a, and 7b, respectively, with minor amounts of the
3-alcohols 3 and 8.5 The radical rearrangement products of each
probe (5 and 10) formed in substantial yield, representing 62% of
the product in the case of 1. The lifetimes of the radical
intermediates formed during the oxidation of 1 (11 ns) and 6 (18
ns) are in close agreement despite a 10-fold difference in inherent
rearrangement rates of the radicals formed (2 × 108 and 2.9 × 107
s-1, respectively). This strongly supports similar radical rebound
mechanisms. No detectable product from cation rearrangement was
observed for 1, while the minor amount from 6 (9) mirrors that
observed in the Barton ester radical abstraction control reactions;
thus, it is attributed to radical rearrangement through internal bond
cleavage. No desaturation products were observed.
a This product elutes in a position that is not fully resolved from that of
the 2-endo product, so a small amount may be produced.
Past studies have shown that the stoichiometrically reduced NDO
component alone can turn over once to yield cis-dihydrodiol
products.3a As shown in Table 1, reduced NDO is also capable of
promoting a single turnover (STO) monooxygenase reaction of 1.
The distribution of products of the reaction is similar to that
observed during multiple turnover of NDOS. The product from
rearrangement of a radical intermediate (5) is observed in 69% yield,
and there is no evidence for a cation intermediate or desaturation
products. This STO reaction shows that the products do not arise
from secondary oxidation reactions.
Mechanistic theory for the Rieske dioxygenases has been
advanced recently by several observations. First, X-ray crystal-
lography has revealed that NDO forms a side-on bound Fe(III)-
hydroperoxy substrate intermediate at the mononuclear iron site.6
Second, the STO experiments have shown that the chemical reaction
can be carried out without NDR and NDF. Both metal centers of
NDO are oxidized during the STO, revealing the sources of the
two reducing equivalents required by the stoichiometry of the
reaction.3a,b This stoichiometry shows that the reactive species is
at the oxidation level of the observed Fe(III)-hydroperoxy species.
Third, it has been shown that the resting, oxidized NDO can also
catalyze the reaction if supplied with H2O2, which contributes both
of the oxygen atoms and the two required electrons.3c This
observation also supports the proposal that the Fe(III)-hydroperoxy
species is a key intermediate. Finally, the first iron chelate
biomimetic complexes have been synthesized that carry out cis-
dihydroxylation reactions.7a,b
Computational studies based on the observations listed above
have revealed an extensive set of possible oxygen insertion
mechanisms.1d,3h,i,7c It was concluded that protonation of the initially
formed peroxy adduct is required to form a reactive species, but
from this point, many paths are possible, as shown in Scheme 1.1d,3h,i
If the reactive species is the Fe(III)-hydroperoxy species itself,
§ University of Minnesota.
‡ Bates College.
¶ Princeton University.
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J. AM. CHEM. SOC. 2007, 129, 3514-3515
10.1021/ja068188v CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Scheme 1. Mechanisms Proposed for Rieske Dioxygenases
unify the results of the enzyme and cis-diol forming biomimetic
compound studies. The reactions of the latter are thought to proceed
through a high-valent intermediate based on the pattern of 18O
solvent exchange.7a,b Early 18O studies showed that O from O2 is
incorporated with high fidelity in the dioxygenase reaction,11 but
some exchange has been noted in the peroxide shunt reaction.3c
Thus, O-O bond cleavage may precede oxygen insertion as
expected for the high-valent intermediate.
Acknowledgment. We thank NIH GM24689 (J.D.L.), NSF
CHE-0221978 (R.N.A., J.T.G.) through the Environmental Mo-
lecular Sciences Institute CEBIC at Princeton University, NSF
CHE-0116233 (R.N.A.), the Camille and Henry Dreyfus Foundation
(R.N.A.), NIH GM072506 (R.N.A.), and NIH GM 036298 (J.T.G.).
Supporting Information Available: Additional methods and
characterization of products (PDF). This material is available free of
charge via the Internet at http://pubs.acs.org.
References
Scheme 2. Mechanism of Norcarane Oxygenation by NDO
(1) (a) Gibson, D. T.; Cruden, D. L.; Haddock, J. D.; Zylstra, G. J.; Brand,
J. M. J. Bacteriol. 1993, 175, 4561-4564. (b) Resnick, S. M.; Lee, K.;
Gibson, D. T. J. Ind. Microbiol. 1996, 17, 438-457. (c) Gibson, D. T.;
Parales, R. E. Curr. Opin. Biotechnol. 2000, 11, 236-243. (d) Boyd, D.
R.; Bugg, T. D. H. Org. Biomol. Chem. 2006, 4, 181-192.
(2) Wackett, L. P.; Kwart, L. D.; Gibson, D. T. Biochemistry 1988, 27, 1360-
1367.
(3) (a) Wolfe, M. D.; Parales, J. V.; Gibson, D. T.; Lipscomb, J. D. J. Biol.
Chem. 2001, 276, 1945-1953. (b) Wolfe, M. D.; Altier, D. J.; Stubna,
A.; Popescu, C. V.; Mu¨nck, E.; Lipscomb, J. D. Biochemistry 2002, 41,
9611-9626. (c) Wolfe, M. D.; Lipscomb, J. D. J. Biol. Chem. 2003, 278,
829-835. (d) Tarasev, M.; Rhames, F.; Ballou, D. P. Biochemistry 2004,
43, 12799-12808. (e) Tarasev, M.; Ballou, D. P. Biochemistry 2005, 44,
6197-6207. (f) Yang, T.-C.; Wolfe, M. D.; Neibergall, M. B.;
Mekmouche, Y.; Lipscomb, J. D.; Hoffman, B. M. J. Am. Chem. Soc.
2003, 125, 7056-7066. (g) Boyd, D. R.; Sharma, N. D.; Bowers, N. I.;
Boyle, R.; Harrison, J. S.; Lee, K.; Bugg, T. D. H.; Gibson, D. T. Org.
Biomol. Chem. 2003, 1, 1298-1307. (h) Bassan, A.; Blomberg, M. R.
A.; Borowski, T.; Siegbahn, P. E. M. J. Phys. Chem. B 2004, 108, 13031-
13041. (i) Bassan, A.; Blomberg, M. R. A.; Siegbahn, P. E. M. J. Biol.
Inorg. Chem. 2004, 9, 439-452. (j) Yu, C. L.; Parales, R. E.; Gibson, D.
T. J. Ind. Microbiol. Biotechnol. 2001, 27, 94-103.
(4) (a) Groves, J. T.; Kruper, W. J.; Haushalter, R. C. J. Am. Chem. Soc.
1980, 102, 6375-6377. (b) Austin, R. N.; Chang, H.-K.; Zylstra, G. J.;
Groves, J. T. J. Am. Chem. Soc. 2000, 122, 11747-11748. (c) Austin, R.
N.; Deng, D.; Jiang, Y.; Luddy, K.; van Beilen, J. B.; Ortiz de Montellano,
P. R.; Groves, J. T. Angew. Chem., Int. Ed. 2006, 45, 8192-8194.
(5) NDO (25 µM), NDR (25 µM), and NDF (250 µM) were reacted with 1
or 6 plus NADH (300 µM) in 0.05 M MES buffer, pH 6.9 plus 0.1 M
NaCl under an air atmosphere at 23 °C for 30 min. 1 and 6 are turned
over by NDOS at about 1/6 the rate of naphthalene at saturation when
determined by O2 uptake. Complete methods are given in the Supporting
Information. In the absence of NDO or NADH, no product was detected.
(6) Karlsson, A.; Parales, J. V.; Parales, R. E.; Gibson, D. T.; Eklund, H.;
Ramaswamy, S. Science 2003, 299, 1039-1042.
the most likely intermediate is a cation after an initial concerted
O-O bond cleavage and oxygen insertion step. Alternatively, if
the O-O bond cleavage occurs as a precursor to substrate attack
to yield an Fe(V)-oxo-hydroxo species, either cationic or radical
intermediates could ensue.
Like the dioxygenase reactions, the monooxygenase reactions
could be envisioned to proceed either directly from the Fe(III)-
hydroperoxy species or after formation of a high-valent Fe(V)-
oxo-hydroxo species. As shown in Scheme 2, if an intermediate
forms, it is likely to be a cation for the Fe(III)-hydroperoxy species
and a radical for the high-valent Fe(V)-oxo-hydroxo species. This
point has been widely discussed in recent monooxygenase litera-
ture.8 As for MMO, concerted addition or the occurrence of very
short-lived intermediates is also possible.9
Our results unequivocally show that monooxygenation reactions
of two alicyclic probes by NDO occur via radical intermediates,
supporting the formation of a high-valent intermediate before the
insertion reaction.10 The lifetime of the radical is comparable to
those observed for many non-heme monooxygenases, such as the
long-chain hydrocarbon oxidizing AlkB family, but much longer
than those observed for P450 and MMO.4c Also, in contrast to the
reactions of the latter two enzymes, the NDO-catalyzed reaction
yields little or no cation-derived ring expansion products, suggesting
that the high-valent intermediate formed is unlikely to abstract a
second e- from the radical intermediate (Scheme 2).
It remains unclear whether the dioxygenation reaction also
proceeds through the high-valent species. However, the apparent
ability of the enzyme to form this species shows that its participation
is possible. This is contrary to the computational studies that
concluded that the energy for formation of high-valent species is
slightly too high for it to materially participate in the dioxygenation
reaction.3h,i Reaction through the high-valent intermediate would
(7) (a) Chen, K.; Costas, M.; Kim, J.; Tipton, A. K.; Que, L., Jr. J. Am. Chem.
Soc. 2002, 124, 3026-3035. (b) Ryu, J. Y.; Kim, J.; Costas, M.; Chen,
K.; Nam, W.; Que, L., Jr. Chem. Commun. 2002, 1288-1289. (c) Bassan,
A.; Blomberg, M. R. A.; Siegbahn, P. E. M.; Que, L., Jr. Angew. Chem.,
Int. Ed. 2005, 44, 2939-2941. (d) Neese, F.; Solomon, E. I. J. Am. Chem.
Soc. 1998, 120, 12829-12848.
(8) (a) Vaz, A. D. N.; McGinnity, D. F.; Coon, M. J. Proc. Natl. Acad. Sci.
U.S.A. 1998, 95, 3555-3560. (b) Newcomb, M.; Shen, R.; Choi, S.-Y.;
Toy, P. H.; Hollenberg, P. F.; Vaz, A. D. N.; Coon, M. J. J. Am. Chem.
Soc. 2000, 122, 2677-2686. (c) Jin, S.; Makris, T. M.; Bryson, T. A.;
Sligar, S. G.; Dawson, J. H. J. Am. Chem. Soc. 2003, 125, 3406-3407.
(d) Newcomb, M.; Shen, R.; Lu, Y.; Coon, M. J.; Hollenberg, P. F.; Kopp,
D. A.; Lippard, S. J. J. Am. Chem. Soc. 2002, 124, 6879-6886. (e) Groves,
J. T. J. Inorg. Biochem. 2006, 100, 434-447.
(9) Gherman, B. F.; Lippard, S. J.; Friesner, R. A. J. Am. Chem. Soc. 2005,
127, 1025-1037.
(10) This conclusion is in accord with evidence for the formation of either a
radical or a cation intermediate in toluene dioxygenase from deuterium
scrambling during oxidation of labeled substrates. Boyd, D. R.; Sharma,
N. D.; Bowers, N. I.; Boyle, R.; Harrison, J. S.; Lee, K.; Bugg, T. D. H.;
Gibson, D. T. Org. Biomol. Chem. 2003, 1, 1298-1307.
(11) Jeffrey, A. M.; Yeh, H. J.; Jerina, D. M.; Patel, T. R.; Davey, J. F.; Gibson,
D. T. Biochemistry 1975, 14, 575-584.
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