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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Am Chem Soc. Author manuscript; available in PMC Aug 4, 2009.
Published in final edited form as:
PMCID: PMC2720596

Radical Intermediates in Monooxygenase Reactions of Rieske Dioxygenases


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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)-dihydroxy-1,2-dihydronaphthalene. NDOS is composed of three proteins: a reductase, a ferredoxin, and an α3β3 oxygenase (NDO). In each α subunit, NDO contains a Rieske Fe2S2 cluster and a mononuclear iron site where substrate dihydroxylation occurs. NDOS also catalyzes monooxygenase reactions for many substrates. The mechanism of the reaction is unknown for either the mono- or di-oxygenase reactions, but has been postulated to involve either direct reaction of a structurally characterized Fe(III)-hydroperoxy intermediate or the electronically equivalent Fe(V)-oxo-hydroxo intermediate formed by O-O bond cleavage before reaction with substrate. The reaction for the former intermediate is expected to proceed through cationic intermediates while the latter is anticipated to initially form a radical intermediate. Here the monooxygenation reactions of the diagnostic probe molecules norcarane and bicyclohexane are investigated. In each case, a significant amount of the rearrangement product derived from a radical intermediate (lifetime of 11–18 ns) is observed while little or no ring expansion product from a cationic intermediate is formed. Thus, monooxygenation of these molecules appears to proceed via the Fe(V)-oxo-hydroxo intermediate. The formation of this high-valent intermediate shows that it must also be considered as a possible participant in the dioxygenation reaction, in contrast to computational studies but in accord with previous biomimetic studies.

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 α3β3 oxygenase (NDO) containing a Fe2S2 Rieske cluster and a mononuclear iron center in each α subunit. NDOS exhibits a broad substrate range for aromatic cis-dihydroxylation.1c Moreover, it catalyzes monooxygenase 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 mono-oxygenation reactions. Concerted 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.

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.

Table 1
Product Distribution from Probe Oxidation by NDOS

Mechanistic theory for the Rieske dioxygenases has been advanced recently by several observations. First, X-ray crystallography 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 2 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, 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.

Scheme 1
Mechanisms Proposed for Rieske Dioxygenases

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 literature.8 For MMO, concerted addition or the occurrence of very short-lived intermediates are also possible.9

Scheme 2
Mechanism of Norcarane Oxygenation by NDO

Our results unequivocally show that monooxygenation reactions of two alicylic 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 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.

Supplementary Material

Supporting Information


We thank NIH GM24689 (JDL), NSF CHE-0221978 (RNA, JTG) through the Environmental Molecular Sciences Institute CEBIC at Princeton University, NSF CHE-0116233 (RNA), the Camille and Henry Dreyfus Foundation (RNA), NIH GM072506 (RNA), and NIH GM 036298 (JTG).


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.


1. (a) Gibson DT, Cruden DL, Haddock JD, Zylstra GJ, Brand JM. J. Bacteriol. 1993;175:4561–4564. [PubMed] (b) Resnick SM, Lee K, Gibson DT. J. Ind. Microbiol. 1996;17:438–457. (c) Gibson DT, Parales RE. Curr. Opin. Biotechnol. 2000;11:236–243. [PubMed] (d) Boyd DR, Bugg TDH. Org. Biomol. Chem. 2006;4:181–192. [PubMed]
2. Wackett LP, Kwart LD, Gibson DT. Biochemistry. 1988;27:1360–1367. [PubMed]
3. (a) Wolfe MD, Parales JV, Gibson DT, Lipscomb JD. J. Biol. Chem. 2001;276:1945–1953. [PubMed] (b) Wolfe MD, Altier DJ, Stubna A, Popescu CV, Münck E, Lipscomb JD. Biochemistry. 2002;41:9611–9626. [PubMed] (c) Wolfe MD, Lipscomb JD. J. Biol. Chem. 2003;278:829–835. [PubMed] (d) Tarasev M, Rhames F, Ballou DP. Biochemistry. 2004;43:12799–12808. [PubMed] (e) Tarasev M, Ballou DP. Biochemistry. 2005;44:6197–6207. [PubMed] (f) Yang T-C, Wolfe MD, Neibergall MB, Mekmouche Y, Lipscomb JD, Hoffman BM. J. Am. Chem. Soc. 2003;125:7056–7066. [PubMed] (g) Boyd DR, Sharma ND, Bowers NI, Boyle R, Harrison JS, Lee K, Bugg TDH, Gibson DT. Org. Biomol. Chem. 2003;1:1298–1307. [PubMed] (h) Bassan A, Blomberg MRA, Borowski T, Siegbahn PEM. J. Phys. Chem. B. 2004;108:13031–13041. (i) Bassan A, Blomberg MRA, Siegbahn PEM. J. Biol. Inorg. Chem. 2004;9:439–452. [PubMed] (j) Yu CL, Parales RE, Gibson DT. J. Ind. Microbiol. Biotechnol. 2001;27:94–103. [PubMed]
4. (a) Groves JT, Kruper WJ, Haushalter RC. J. Am. Chem. Soc. 1980;102:6375–6377. (b) Austin RN, Chang H-K, Zylstra GJ, Groves JT. J. Am. Chem. Soc. 2000;122:11747–11748. (c) Austin RN, Deng D, Jiang Y, Luddy K, van Beilen JB, Ortiz de Montellano PR, Groves JT. Angew. Chem. Int. Ed. 2006;45:8192–8194. [PubMed]
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 Supplemental Information. In the absence of NDO or NADH, no product was detected.
6. Karlsson A, Parales JV, Parales RE, Gibson DT, Eklund H, Ramaswamy S. Science. 2003;299:1039–1042. [PubMed]
7. (a) Chen K, Costas M, Kim J, Tipton AK, Que L., Jr J. Am. Chem. Soc. 2002;124:3026–3035. [PubMed] (b) Ryu JY, Kim J, Costas M, Chen K, Nam W, Que L., Jr Chem. Commun. 2002:1288–1289. [PubMed] (c) Bassan A, Blomberg MRA, Siegbahn PEM, Que L., Jr Angew. Chem. Int. Ed. 2005;44:2939–2941. [PubMed] (d) Neese F, Solomon EI. J. Am. Chem. Soc. 1998;120:12829–12848.
8. (a) Vaz ADN, McGinnity DF, Coon MJ. Proc. Natl. Acad. Sci. U. S. A. 1998;95:3555–3560. [PubMed] (b) Newcomb M, Shen R, Choi S-Y, Toy PH, Hollenberg PF, Vaz ADN, Coon MJ. J. Am. Chem. Soc. 2000;122:2677–2686. (c) Jin S, Makris TM, Bryson TA, Sligar SG, Dawson JH. J. Am. Chem. Soc. 2003;125:3406–3407. [PubMed] (d) Newcomb M, Shen R, Lu Y, Coon MJ, Hollenberg PF, Kopp DA, Lippard SJ. J. Am. Chem. Soc. 2002;124:6879–6886. [PubMed] (e) Groves JTJ. Inorg. Biochem. 2006;100:434–447. [PubMed]
9. Gherman BF, Lippard SJ, Friesner RA. J. Am. Chem. Soc. 2005;127:1025–1037. [PubMed]
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 DR, Sharma ND, Bowers NI, Boyle R, Harrison JS, Lee K, Bugg TDH, Gibson DT. Org. Biomol. Chem. 2003;1:1298–1307. [PubMed]
11. Jeffrey AM, Yeh HJ, Jerina DM, Patel TR, Davey JF, Gibson DT. Biochemistry. 1975;14:575–584. [PubMed]
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