Dioxygenase Enzymes: Catalytic Mechanisms and Chemical Models

Tetrahedron 59 (2003) 7075–7101

Tetrahedron report number 648 Dioxygenase enzymes: catalytic mechanisms and chemical models

Timothy D. H. Bugg* Department of Chemistry, University of Warwick, Coventry CV4 7AL, UK

Received 10 June 2003

Contents 1. Introduction 7075 1.1. Di-oxygenase and mono-oxygenase enzymes 7075 1.2. Oxygen activation by metal ions 7076 1.3. Reactivity of alkyl hydroperoxide intermediates 7077 2. Catechol dioxygenases 7078 2.1. Intradiol catechol dioxygenases 7079 2.2. Model chemistry for intradiol catechol cleavage 7080 2.3. Extradiol catechol dioxygenases 7082 2.4. Model chemistry for extradiol catechol cleavage 7084 3. Arene (dihydroxylating) dioxygenases 7086 3.1. Napthalene dioxygenase: structure and catalytic mechanism 7086 3.2. Model reactions for non-heme iron-catalysed cis-dihydroxylation 7087 4. a-Ketoglutarate-dependent dioxygenases 7088 4.1. Enzymology of a-ketoglutarate-dependent dioxygenases 7088 4.2. Model chemistry for a-ketoglutarate-dependent hydroxylation 7090 5. Lipoxygenase and cyclo-oxygenase 7090 5.1. Lipoxygenases 7090 5.2. Cyclo-oxygenase 7091 5.3. Model chemistry for hydrogen atom abstraction 7091 6. Other metal-dependent dioxygenases 7092 6.1. Indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase 7092 6.2. Quercetin 2,3-dioxygenase 7093 6.3. b-Carotene dioxygenase 7094 6.4. Methionine salvage pathway dioxygenases 7094 6.5. Acetylacetone dioxygenase 7095 6.6. Cysteine dioxygenase 7095 7. Cofactor-independent dioxygenases 7095 7.1. Epoxyquinone natural product biosynthesis in Streptomyces 7096 7.2. Oxidative cleavage of 3-hydroxy-4-oxoquinolines by bacterial 2,4-dioxygenases 7096 7.3. Other cofactor-independent dioxygenases 7097

1. Introduction hence there is considerable interest in elucidating the catalytic mechanism of these enzymes. This review will A number of enzymes found in Nature are able to catalyse describe the current understanding of the catalytic mechan- the activation of dioxygen from the atmosphere, and use it to ism of a number of dioxygenase-catalysed reactions, and effect a wide variety of remarkable reactions. Many of these how in several cases this knowledge has been harnessed to reactions have little or no precedent in organic chemistry, design catalysts for biomimetic model chemistry.

Keywords: dioxygenase; metalloenzyme; biomimetic; model chemistry. 1.1. Di-oxygenase and mono-oxygenase enzymes * Tel.: þ44-2476-573018; fax: þ44-2476-524112; e-mail: [email protected] Enzymes that are able to activate dioxygen are divided into

0040–4020/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0040-4020(03)00944-X 7076 T. D. H. Bugg / Tetrahedron 59 (2003) 7075–7101 oxidases, which use oxygen as an oxidant, and reduce Orbital overlap with a metal ion. Upon complexation of dioxygen to hydrogen peroxide or water; and oxygenases, dioxygen to a transition metal ion containing unpaired 3d which incorporate oxygen atoms from dioxygen into the electrons, the unpaired electrons in the dioxygen p* orbitals product(s). Mono-oxygenases catalyse the incorporation of are able to overlap with those on the metal ion.4 The one atom of oxygen into the product, while di-oxygenases reaction of such a transition metal-dioxygen with a singlet incorporate both atoms of oxygen into the product(s). organic reagent is then allowed, provided that the overall number of unpaired electrons in the complex remains Most dioxygenase enzymes require a metal cofactor (for constant. reasons to be discussed below), which is most often iron(II) or iron(III). This review will concentrate on families of non- Single electron transfer. The transition metals found in heme iron(II)-dependent dioxygenases,1 for which recent metallo-enzymes that activate dioxygen have two consecu- model chemistry is available. Section 6 will mention one tive available oxidation states (e.g. Fe(II)/Fe(III), Cu(I)/- heme-dependent dioxygenase,2 and Section 7 will describe Cu(II)), hence the metal centre is able to carry out single 3 a small number of cofactor-independent dioxygenases that electron transfer to bound dioxygen. The O2 oxygen have recently been discovered. ground state can accept a single electron to form super- oxide—an allowed reaction—which is one possible route 1.2. Oxygen activation by metal ions for oxygen activation. Superoxide can then participate in a variety of 1- or 2-electron chemical reactions.5,6 Reactions involving the oxidation of hydrocarbons by dioxygen are exothermic, since the sum of the bond A criticism of this route is that it is apparently energetically 2 energies of the C–O and O–H bonds formed is greater unfavourable. The reduction potential for the O2/O2 couple than the sum of the bond energies of the C–C, C–H, and is 20.16 V in water at pH 7.0.6 The reduction potential for O–O bonds broken.3 In spite of this exothermicity, the Fe(III)/Fe(II) couple is þ0.77 V. Therefore, the redox dioxygen is chemically unreactive in the absence of a potential for the activation of dioxygen to superoxide by suitable catalyst. The reason is that the ground state for Fe(II) is 20.93 V. However, the redox potentials for the 3 dioxygen ( O2) contains two unpaired electrons in the Fe(II)/Fe(III) and dioxygen/superoxide couples in a metal- highest occupied p* orbitals, and is therefore spin- loenzyme would be dependent upon the particular micro- forbidden to react with spin-paired singlet species. In environment of the active site. It is known that redox 1 contrast, the singlet excited state of dioxygen ( O2), which enzymes can strongly influence the redox potential of contains a pair of valence electrons, is highly reactive cofactors bound at their active sites, by selective stabilis- towards alkenes and dienes, as shown in Figure 1. ation of either the oxidised or the reduced form.7 Thus, for 1 However, since O2 is 22 kcal/mol higher in energy than example, if the enzyme formed stabilising interactions with 3 O2, it is not feasible for oxygenase enzymes to access this bound superoxide but not dioxygen (e.g. electrostatic excited state. interaction, hydrogen bonding, or protonation), then the reaction would be favoured. Activation of dioxygen to Transition metal ions containing unpaired electrons can use superoxide has been observed in hemoglobin,8 and in three strategies to activate dioxygen. EDTA–Fe(III) complexes.9 Therefore, it is feasible that this route can be used by metalloenzymes, and there is some evidence that it takes place in the extradiol catechol dioxygenases.

Single electron transfer to dioxygen can also take place from organic reagents which can access a stable radical intermediate. Thus, the reduced flavin cofactor found in

3 1 Figure 1. (A) Molecular orbital diagrams for O2, O2. (B) Reactions of 1 O2. Figure 2. Activation of dioxygen by reduced flavin. T. D. H. Bugg / Tetrahedron 59 (2003) 7075–7101 7077 flavoprotein hydroxylase enzymes is able to activate 1.3. Reactivity of alkyl hydroperoxide intermediates dioxygen via a single electron transfer to form a stable flavin semiquinone and superoxide, which then recombine Since dioxygenase enzymes catalyse the incorporation of to form a hydroperoxide intermediate (Fig. 2).10 two atoms of oxygen from dioxygen into the products of the reaction, a common strategy is to form one C–O bond with Reaction with a substrate radical. Since the reaction of the substrate first, before O–O bond cleavage, in the form of dioxygen via radical mechanisms is a spin-allowed process, an alkyl hydroperoxide intermediate. Organic hydro- reaction with a substrate radical is an alternative possible peroxides are chemically reactive, due to the weakness of mechanism. It has been proposed that a substrate activation the O–O bond, and can react in several different ways: as a mechanism of this kind occurs in the intradiol catechol nucleophile, as an electrophile, O–O cleavage via C–C dioxygenases, where a bound catechol semiquinone inter- bond migration, or O–O cleavage via homolytic cleavage. mediate attacks dioxygen to form a hydroperoxide radical Examples of each type of reactivity will be given, in order to (Fig. 3).1 illustrate the variety of possible reaction pathways.

Nucleophilic reactivity. In common with other reagents containing adjacent heteroatoms (e.g. N2H4,NH2OH), hydroperoxides possess a high degree of nucleophilicity. Intermediates containing an adjacent carbonyl group may undergo nucleophilic attack to form a dioxetane intermedi- ate. One biological example of this reactivity occurs in the firefly luciferase enzyme, where a dioxetane intermediate is formed, and then decays to release an excited state of the product, which releases luminescence (Fig. 4(A)).11 This type of reactivity is also found in the mechanism of action of the vitamin K-dependent carboxylase, where a vitamin K hydroperoxide carries out a conjugate addition on the adjacent alkene, and the subsequent dioxetane is opened to 12 Figure 3. Substrate activation mechanism proposed for intradiol catechol form an epoxide (Fig. 4(B)). Base catalysis is required in cleavage.

Figure 4. Dioxetane intermediates in reactions catalysed by (A) firefly luciferase (B) vitamin K-dependent carboxylase. 7078 T. D. H. Bugg / Tetrahedron 59 (2003) 7075–7101 these cases, in order to generate the nucleophilic hydroper- oxide anion.

Electrophilic reactivity. Cleavage of the O–O bond of a hydroperoxide can occur via the attack of a nucleophile, in which case the hydroperoxide acts as an oxidising agent. One biologically important example is the flavin 4a- hydroperoxide intermediate formed in flavoprotein hydro- xylases. In the case of p-hydroxybenzoate hydroxylase, the flavin hydroperoxide reacts with a phenol nucleophile, resulting in hydroxylation of the aromatic ring (Fig. 5).10

Figure 6. Criegee rearrangements of alkyl hydroperoxides.

Figure 5. Reaction of flavin hydroperoxide as an electrophile in p-hydroxybenzoate hydroxylase.

O–O Cleavage via C–C bond migration. Upon acid catalysis, the migration of a C–C bond onto an electron- deficient oxygen can occur. In alkyl hydroperoxides, this reaction is known as a Criegee rearrangement (Fig. 6).13 This type of 1,2-rearrangement occurs in the Baeyer– Figure 7. Homolytic O–O cleavage of organic hydroperoxides. Villiger oxidation of ketones by hydrogen peroxide and 14 peracids. While substituent effects have a strong influence The reader will see that there are a number of possible types on the migratory aptitude of migrating groups in these of reaction that alkyl hydroperoxides can undergo, and reactions, recent studies by Goodman and Kishi have also hence there are several different mechanistic possibilities shown that stereoelectronic factors can determine the for dioxygenase enzyme-catalysed reactions. selectivity of the Criegee rearrangements.15

O–O Cleavage via homolytic cleavage. In the presence of transition metal catalysts, especially iron salts, organic 2. Catechol dioxygenases peroxides can fragment via homolytic cleavage to give an alkoxy radical, which can further fragment via homolytic The catechol dioxygenases catalyse the oxidative cleavage cleavage of an adjacent C–C bond. Examples of such of catechol and substituted catechols, a key step in the reactions are shown in Figure 7.16 –18 Assuming that O–O bacterial degradation of aromatic compounds in the bond cleavage is triggered by complexation to iron(II) in environment.19,20 Two families of dioxygenase enzyme these cases, the other product of homolytic cleavage is an were discovered by Hayaishi which can catalyse the iron(III) oxo complex. High oxidation state iron–oxo oxidative cleavage of catechol, both families utilising species are believed to be catalytic intermediates in dioxygen as a substrate (Fig. 8).21 –23 The intradiol cytochrome P450 and non-heme iron-dependent metallo- dioxygenases, typified by catechol 1,2-dioxygenase (or enzyme-catalysed reactions.2 pyrocatechase), cleave the carbon–carbon bond between T. D. H. Bugg / Tetrahedron 59 (2003) 7075–7101 7079

oligomeric (abFe)12 structure. The non-heme iron(III) cofactor was found to be ligated by four amino acid side- chains: the imidazole side-chains of His-460 and His-462, and the phenolic side-chains of Tyr-408 and Tyr-447 (Fig. 10(A)). A fifth water ligand completes a trigonal bipyr- amidal structure. The two tyrosinate ligands are thought to stabilise the iron(III) cofactor and give the enzyme its characteristic deep red colour due to ligand-to-metal charge transfer interactions,24 which give rise to characteristic Figure 8. Reactions catalysed by intradiol and extradiol dioxygenases. resonance Raman vibrations at 1254 and 1266 cm21.25 The X-ray structures of protocatechuate 3,4-dioxygenase from the phenolic hydroxyl groups to yield muconic acid as the Pseudomonas aeruginosa and Acinetobacter sp. ADP1 have product, and require Fe3þ as a cofactor.21 The extradiol also been solved, revealing very similar active site dioxygenases, typified by catechol 2,3-dioxygenase (or structures.26,27 metapyrocatechase), cleave the carbon–carbon bond adjacent to the phenolic hydroxyl groups to yield The structure of another member of the intradiol dioxygenase 2-hydroxymuconaldehyde as the product, and require Fe2þ family, catechol 1,2-dioxygenase (1,2-CTD) from Acineto- as a cofactor.22 They are therefore a particularly striking bacter sp. ADP1, has also been solved.28 This enzyme class of dioxygenase, since both intradiol and extradiol consists of an a2 homodimer with one iron(III) cofactor per dioxygenases require a mononuclear iron centre with no subunit. The tertiary structure of the 1,2-CTD enzyme is additional cofactors, and the dependence upon iron(II) similar to that found in 3,4-PCD, although 1,2-CTD versus iron(III) is intriguing. contains a novel helical zipper motif at the interface of the two subunits, and contains two molecules of bound 2.1. Intradiol catechol dioxygenases phospholipid. The active site of 1,2-CTD contains a very similar arrangement of iron(III) ligands: Tyr-200 and His- 18 Hayaishi was able to demonstrate, using O2 labelling 226 are the axial ligands, and Tyr-164, His-224 and a water experiments, that catechol 1,2-dioxygenase from Pseudo- molecules are the equatorial ligands (Fig. 10(C)).28 monas incorporated both atoms of oxygen from dioxygen into the reaction products.21 The mechanism invoked by Structures of 3,4-PCD with bound catechol substrates reveal Hayaishi to explain these results was that a four-membered that, upon substrate binding, the axial tyrosine ligand Tyr- dioxetane ring was formed during the reaction (Fig. 9), 447 and equatorial water ligand are both displaced, to form a which fragmented to form the reaction products.21 monodentate substrate complex.29,30 Tyr-447 swings away from the iron(III) cofactor to leave an iron(III) centre with Although the catechol dioxygenases were demonstrated by approximately octahedral geometry, but containing a vacant Hayaishi to be crystalline enzymes,23 it was not until 1988 co-ordination site opposite His-460. Subsequent structures that the first X-ray structure of a catechol dioxygenase, the of 3,4-PCD with substrates and inhibitors in the presence of intradiol-cleaving protocatechuate 3,4-dioxygenase (3,4- NO and CN2 have revealed that either NO or CN2 can bind PCD) from Pseudomonas putida, was solved by Ohlendorf in the vacant co-ordination site, to form a ternary et al.24 The enzyme consists of two subunits, in an complex.30 – 32 The structures of 1,2-CTD with bound

Figure 9. Mechanism involving a dioxetane intermediate.

Figure 10. Active site structures of intradiol catechol dioxygenases: (A) protocatechuate 3,4-dioxygenase from Pseudomonas putida; (B) 3,4-PCD in complex with substrate; (C) catechol 1,2-dioxygenase from Acinetobacter sp. ADP1. 7080 T. D. H. Bugg / Tetrahedron 59 (2003) 7075–7101 catechol have shown, similarly, that the axial Tyr-200 more efficient hydrolysis of the anhydride intermediate.33 ligand swings away from the iron(III) centre upon substrate Model reactions for intradiol cleavage (see below) have in binding. some cases yielded muconic anhydrides as the reaction products, again consistent with a 1,2-rearrangement. Reaction of the iron(III)–catechol complex with dioxygen has been proposed to occur via a substrate activation Hence, the overall reaction involves activation of the mechanism, in which the bound substrate has significant substrate, via a semiquinone intermediate, for reaction iron(II)–semiquinone character, and is hence able to react directly with dioxygen, followed by rearrangement of a directly with dioxygen to form a hydroperoxide intermedi- hydroperoxide intermediate by acyl migration. The role of ate. The reasons for this proposal are that no change in the the axial Tyr-447 ligand that swings away from the iron(III) iron(III) EPR signal is detectable during the catalytic cycle, centre during catalysis has been probed by construction of a and that model systems showing the highest reactivity Y447H mutant 3,4-PCD enzyme.32 The mutant enzyme has (Section 2.2) are those in which the substrate complex a 600-fold lower kcat than the native enzyme, but kinetic shows more iron(II)–semiquinone character. studies have shown that reaction with dioxygen occurs at similar rates to the native enzyme, therefore the lower kcat is Subsequent reaction of the cyclohexadienyl hydroperoxide due to slower substrate binding and product release.32 The intermediate is believed to occur via migration of the role of Tyr-447 is thought to be as a base, deprotonating the adjacent acyl group (acyl migration) to yield muconic second phenolic hydroxyl group of the substrate to form a anhydride as an intermediate, which undergoes hydrolysis catechol dianion. The proposed mechanism is shown in 18 to give the product muconic acid. O2 labelling studies on Figure 12. catechol 1,2-dioxygenase from Pseudomonas arvilla have revealed that the intradiol cleavage products contain 99% 2.2. Model chemistry for intradiol catechol cleavage incorporation of a single atom of 18O, and 74% incorpor- ation of a second atom of 18O, with 24% incorporation of A number of iron(III)-based model complexes have been only one atom of 18O(Fig. 11).33 These data are not found to effect intradiol catechol cleavage to yield muconic consistent with a dioxetane intermediate, but could be anhydride products, or furanone derivatives of the muconic explained by a Criegee rearrangement to give an anhydride acid product. A comprehensive review of these iron(III) intermediate, followed by the partial exchange of the models has recently been published,34 and thus I will discuss iron(III) 18O–hydroxide with solvent water.33 Interestingly, only a few selected examples. no single atom isotope incorporation is observed with the natural substrate catechol, implying a lack of exchange or a The first model system for intradiol cleavage was an Fe(III)–nitrilotriacetate (NTA) complex 1,whichwas reported to convert 3,5-di-tert-butylcatechol catalytically over a period of four days in the presence of oxygen to give the furanone derivative 2 in 80% yield.35,36 An X-ray crystal structure of this complex showed the catechol substrate bound in bidentate fashion, with the geometry around the central Fe(III) close to octahedral.36 Labelling studies with 18 O2 on this system revealed the incorporation of one atom 18 of O2 into the furanone 2, consistent with the existence of an anhydride intermediate as shown in Figure 13.36

Subsequent studies using a range of iron(III) complexes

18 showed a correlation between the reactivity of the Fe(III)– Figure 11. O2 Labelling studies on the reaction of pyrogallol with ligand system and the Lewis acidity of the metal centre, catechol 1,2-dioxygenase from Pseudomonas arvilla. which could be quantitatively assessed by measuring the

Figure 12. Catalytic mechanism for intradiol dioxygenases. T. D. H. Bugg / Tetrahedron 59 (2003) 7075–7101 7081 reaction with dioxygen. The order of reactivity of the coordinated 3,5-di-tert-butylcatecholate ligand (dbc)22 was in the order [Fe(3)(dbc)]þ.[Fe(4)(dbc)]þ. [Fe(5)(dbc)]þ.[Fe(6)(dbc)]þ, and is correlated to the Lewis acidity of the iron(III) centre.39

Figure 13. Intradiol cleavage catalysed by Fe(III)–NTA complex 1.R¼H, Me. The reactivity of a series of Fe(III) complexes of tetradentate ligands 7–9 with 3,5-di-tert-butylcatechol, which all form [Fe(L)(dbsq)] complexes with significant semiquinone (sq) character, was found to correlate inversely with the lmax value, the order of reactivity being 7 (935 nm).8 (941 nm).9 (957 nm).40 The position of the ligand–metal charge-transfer (LMCT) bands for Fe(III) complexes of 10 was observed to shift to lower energies by varying the substituents on the catecholate from electron- withdrawing to electron-donating. The reaction with dioxy- gen exhibited pseudo-first-order kinetics, and the order of activity correlated with the energy of the lower energy LMCT band of the complexes, electron-donating substitu- ents on the catechol resulting in a higher dioxygenase activity.41

redox potential for the catechol-to-semiquinone oxidation.37 Of the complexes studied, the FeIII –nitrilotriacetate com- plex 1 showed the highest reactivity, and the highest redox potential of þ59 mV (and hence the highest affinity of the catechol ligand for the Fe(III) centre).37

Further studies by Que and co-workers led to the discovery of more reactive Fe(III) complexes,38,39 the most active of which was Fe(III)–tris(2-pyridylmethyl)amine (TPA) 3. This complex was found to react with dioxygen within minutes to form furanone 2 in 98% yield, at a rate of 15 M21 s21, approximately 1000-fold faster than complex 1. Analysis of complex 3 by X-ray crystallography and 1H NMR spectroscopy revealed a very strong iron–catecho- late interaction, and increased semiquinone character in the bound substrate. It was therefore proposed that A catalytically active model system has been reported by formation of a transient Fe(II)–semiquinone intermediate Kruger et al. using N,N0-dimethyl-2,11-diaza[3,3](2,6)pyr- preceded reaction with dioxygen. Each of these tetra- idinophane (11) as a macrocyclic ligand.42 For catalytic dentate ligands 3–6, when complexed to Fe(III), showed reaction, two equivalents of base were required, per mole of activity for intradiol cleavage, revealing that two easily iron(III). With 1% of the iron(III) catalyst, a yield of 54% of accessible cis coordination sites are needed for coordi- muconic anhydride was obtained after a reaction time of nation of the catecholate ligand and its subsequent 30 h.42 7082 T. D. H. Bugg / Tetrahedron 59 (2003) 7075–7101 The crystal structures of three other extradiol dioxygenases have now been solved. In 1998, Kita et al. reported the structure of catechol 2,3-dioxygenase from Pseudomonas 47 putida mt-2, an a4 tetramer. The subunit structure is very similar to that of BphC, and the iron(II) cofactor is bound by His-153, His-214 and Glu-265.47 In 1999, Sugimoto et al. reported the structure of protocatechuate 4,5-dioxygenase from Sphingomonas paucimobilis SYK-6, which is com- 48 posed of an a2b2 tetramer. This enzyme has no sequence similarity to BphC, yet the disposition of iron(II) ligands is Thus, intradiol cleavage activity is observed with a number very similar: the metal centre is co-ordinated by His-12, of tetradentate nitrogen ligands, complexed to Fe(III), His-61 and Glu-242 (Fig. 14(B)).48 In 2000, Titus et al. mimicking the tetradentate Fe(III) co-ordination state in reported the structure of human homogentisate dioxygen- the intradiol dioxygenase active sites. High activity is ase, a non-heme iron(II)-dependent dioxygenase involved in correlated with Lewis acidity of the Fe(III) centre, and the mammalian degradation of L-phenylalanine and L- semiquinone character of the bound catechol substrate. tyrosine.49 This enzyme bears no sequence similarity to the Reactivity also correlates with the ligand-to-metal charge- bacterial extradiol catechol dioxygenases, yet its active site transfer band of the complex, which is believed to strongly features are very similar: the iron(II) cofactor is bound by influence the electronic structure and reactivity of the His-335, Glu-341 and His-371.49 iron(III) centre in the enzyme active site.43 The catalytic mechanism of the extradiol catechol dioxy- 2.3. Extradiol catechol dioxygenases genases proceeds through several enzyme-bound intermedi- ates. A discussion of the evidence and mechanistic The extradiol catechol dioxygenases catalyse the oxidative possibilities for these enzymes has recently been pub- cleavage of the carbon–carbon bond adjacent to the lished,50 and therefore this section will comment on the key phenolic hydroxyl groups, to give a 2-hydroxymucon- pieces of evidence for each step. aldehyde product, using iron(II) as a cofactor (Fig. 8). It was shown by Hayaishi that catechol 2,3-dioxygenase from EPR spectroscopic studies by Lipscomb and co-workers of Pseudomonas arvilla incorporated two atoms of 18O from the NO complex of the extradiol dioxygenase protocatech- 18 21 O2 into the product, and thus a mechanism involving a uate 4,5-dioxygenase demonstrated that the iron(II) cofactor dioxetane intermediate was also possible for this family of binds both catecholic hydroxyl groups, and binds NO; enzymes. however, no iron(III) intermediates could be detected during turnover.51 Evidence for a semiquinone radical intermediate The structure of 2,3-dihydroxybiphenyl 1,2-dioxygenase has been obtained in the reaction catalysed by 2,3- (BphC) from Pseudomonas LB400, a strain capable of dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) from degrading chlorinated biphenyls, was solved by Han et al. Escherichia coli, using substrate analogues containing in 1996.44 The tertiary structure of the enzyme consists of cyclopropyl radical traps.52 Both trans- and cis-substituted two similar babbb domains, only one of which contains cyclopropyl analogues (12a,b) were found to be efficiently an iron(II) cofactor. A funnel-shaped cavity leads to the processed by the enzyme. However, analysis of the products active site, where the iron(II) centre is ligated by three by further enzymatic degradation revealed that isomerisa- amino acid side-chains: His-146, His-210 and Glu-260 tion of the cyclopropyl ring substituents had occurred: 44 (Fig. 14). This His2Glu/Asp motif is found in a number processing of the trans-substituted substrate gave 94% of other non-heme iron(II)-dependent oxygenases, includ- trans-product and 6% cis-product, while processing of the ing the a-ketoglutarate dependent dioxygenases and cis-substituted substrate gave only 10% of the cis-product, isopenicillin N synthase.45 The crystal structure of and 90% of the trans-product (Fig. 15). The most plausible BphC from Pseudomonas KKS102 has also been solved explanation of these results is that a radical-mediated with bound substrate, yielding a similar co-ordination reversible opening of the cyclopropyl ring is taking place, geometry, however, the crystallised form of the enzyme upon formation of a transient iron(II)–semiquinone-super- contains iron(III) rather than iron(II).46 oxide intermediate.52

Figure 14. Active sites structures of extradiol catechol dioxygenases: (A) 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC) from Pseudomonas sp. LB400; (B) protocatechuate 4.5-dioxygenase from Sphingomonas paucimobilis SYK-6. T. D. H. Bugg / Tetrahedron 59 (2003) 7075–7101 7083

Figure 15. cis–trans Isomerisation of a cyclopropyl radical trap (12) catalysed by extradiol dioxygenase MhpB. Thus, it is believed that the iron(II) cofactor mediates one- electron transfer with dioxygen and with the bound catechol substrate, which has been shown by UV/visible and resonance Raman spectroscopy to be bound as the mono- anion.53 C–O bond formation with the two activated substrates then generates a hydroperoxide intermediate. There are two possible hydroperoxides that could be formed, either proximal or distal to the C–C bond being cleaved. Inspection of models reveals that the geometry required to form the distal hydroperoxide is rather strained, The proximal hydroperoxide is then believed to undergo and inspection of the X-ray structures of BphC also Criegee rearrangement to give a seven membered lactone indicates that reaction at the proximal position of the intermediate, which is hydrolysed to give the extradiol 18 substrate is more readily achievable than reaction at the product. O2 labelling studies carried out on E. coli MhpB distal position.44 A series of ‘carba’ analogues of the revealed that, although both the acid and ketone carbonyls 18 18 18 proximal and distal hydroperoxides were synthesised for could be labelled with O from O2, upon reaction in H2 O MhpB from E. coli, in which the –OOH functional group the carboxylate position was labelled to the extent of 30%, was replaced by –CH2OH, and the cyclohexadienyl ring consistent with the formation of an a-ketolactone inter- simplified to a cyclohexanone ring.54 Carba analogue 13 of mediate, and exchange of iron(II) hydroxide with solvent the distal hydroperoxide showed no inhibition of MhpB at 18O-labelled water (Fig. 16).55 The enzyme was also found 10 mM concentration. However, analogues 14 and 15 of the to catalyse the hydrolysis of a saturated seven-membered proximal hydroperoxide did show modest competitive lactone analogue (16).55 These studies implicate a lactone inhibition of MhpB, with Ki values of 4.9 mM and intermediate arising from Criegee rearrangement in the 0.7 mM, respectively. Enzyme inhibition was only observed extradiol cleavage reaction mechanism. Thus, the extradiol in analogues in which the hydroxymethyl substituent is reaction mechanism involves activation of dioxygen by one- positioned in an axial orientation with respect to the electron transfer, followed by hydroperoxide formation, cyclohexanone ring, indicating that the conformation Criegee rearrangement, and lactone hydrolysis, as shown in adopted by the hydroperoxide was of importance. Figure 16.

Insight into the role of other active site residues in extradiol dioxygenase catalysis has been provided by a recent structure of Pseudomonas KKS102 BphC complexed with 7084 T. D. H. Bugg / Tetrahedron 59 (2003) 7075–7101

Figure 16. Catalytic mechanism for extradiol dioxygenases. substrate and NO. The structure reveals that NO occupies a give the 2-pyrone products 18 and 19 (Fig. 17). Funabiki sixth co-ordination site at the iron(II) centre, positioned et al. found that FeCl3 complexes with bipyridine/pyridine close to His-194, which in turn is positioned close to the C-3 prepared in situ cleave 3,5-di-tert-butylcatechol over 24 h to hydroxyl group of the substrate.56 His-194 is proposed to act give a mixture of products containing 50–70% orthoqui- as a base to deprotonate the substrate at C-3, to form the none 20, 15–30% intradiol products, and 2–5% pyrones 18 18 18 59 catechol monoanion, and then the protonated imidazolium and 19, which incorporate O label from O2. Funabiki side-chain of His-194 is proposed to stabilise the negative proposed that these pyrones are derived from loss of CO charge of bound superoxide, hence assisting oxygen from the a-ketolactone extradiol cleavage intermediate 17, activation. This residue may further act as a proton donor although this decarbonylation reaction would be mechan- to assist the Criegee rearrangement. istically unusual. A mixture containing 17 was incubated under the reaction conditions, and resulted in a slow Although iron(II) is normally utilised as the metal ion increase (over 24–96 h) in levels of 18, however, the slow cofactor in these enzymes, dioxygenase MndD from timescale of this experiment, and the presence of mixtures Arthrobacter globiformis contains manganese(II) at its of compounds, does not convincingly demonstrate that 18 active site.57 A similar catalytic mechanism can be would be formed from 17 during catechol cleavage. envisaged for Mn2þ, although the redox potential for the Nevertheless, pyrones 18 and 19 have been referred to as Mn3þ/Mn2þ redox couple is significantly higher, at ‘extradiol’ in the literature since 1986. þ1.6 V. Gentisate dioxygenase, which catalyses the oxi- dative cleavage of a p-hydroquinone substrate, requires Several complexes of tridentate N3-ligands with iron(III) iron(II), which has been shown to ligate the C-2 hydroxyl and 3,5-di-tert-butylcatechol have been found to yield group of the substrate, and appears to follow a similar pyrones 18 and 19 upon exposure to oxygen. Dei et al. mechanism.58 obtained a 35% yield using complex [FeIII- (TACN)(dbc)]Cl (21),60 while Que et al. used the same 2.4. Model chemistry for extradiol catechol cleavage complex to give an almost quantitative yield in CH2Cl2 in the presence of AgOTf.61 Jo and Que have further The vast majority of metal-based complexes designed for reported that a tridentate ligand that forms a planar, catechol oxidative cleavage perform intradiol cleavage, meridional complex with iron(III) gives no pyrone which therefore appears to be the kinetically favoured product, but instead a mixture of quinone 20 and pathway under most conditions. There are several examples intradiol cleavage products.62 It therefore appears that a of iron complexes that cleave 3,5-di-tert-butylcatechol to facial, tridentate ligand is required for this reaction. We

Figure 17. Production of 2-pyrones. T. D. H. Bugg / Tetrahedron 59 (2003) 7075–7101 7085 have reported that the oxygenation of catechol by O2 in role in the reaction beyond that of acting as a base. In the thepresenceofFeCl2 or FeCl3, 1,4,7-triazacyclononane presence of one equivalent of pyridinium hydrochloride, the (TACN), and pyridine in methanol gives the authentic selectivity of the reaction switches back to a 4:1 extradiol to extradiol product 2-hydroxymuconic semi-aldehyde intradiol ratio, implying a role for the pyridinium ion as a methyl ester 22 in 50% yield, and the intradiol product proton donor, to assist the Criegee rearrangement for 63 monomethyl muconate 23. In this model system, FeCl2/ extradiol cleavage. Cleavage in the presence of methanol, TACN shows higher selectivity for extradiol/intradiol ethanol or isopropanol yields the respective alkyl esters of cleavage (7:1) compared with FeCl3/TACN (2:1), as 2-hydroxymuconic semialdehyde, implying the intermedi- shown in Figure 18, indicating that iron(II) has a acyofasevenmembereda-ketolactone, as for the higher intrinsic preference for extradiol cleavage in this enzymatic reaction.64 reaction than iron(III), as found in the catechol dioxygenases. Extradiol cleavage of catechol has been reported using potassium superoxide in DMSO, giving 2-hydroxymuco- naldehyde in 5–10% yield.66 Further investigation of this reaction by Lin et al. revealed that the rate is largely independent of substituents on the catechol ring, whereas the FeCl2/TACN reaction is unable to cleave catechols bearing electron-withdrawing groups, as found for extradiol 66 dioxygenase MhpB. Given that the KO2 reaction occurs under basic conditions and is independent of substituent, it seems probable that this reaction occurs via a dioxetane intermediate. Extradiol cleavage of 3,5-di-tert-butylcate- chol by FeCl2 or FeCl3 in THF/water mixtures has been reported by Funabiki and co-workers.67 They report 67 increased extradiol selectivity for FeCl2, as observed in 63,64 the FeCl2/TACN model reaction.

In summary, attempts to model the extradiol dioxygenase reaction have shown that the reaction is more difficult to Extradiol cleavage is only observed with the facial N3- achieve, and appears to require a facial tridentate ligand, tridentate ligand TACN, and not with macrocyclic ligands binding of the catechol mono-anion, and the involvement of containing nitrogen and oxygen donors.64 The reaction a proton donor. These observations seem to parallel what is proceeds in the absence of pyridine using monosodium seen in the extradiol dioxygenase active site, and the catecholate, but not disodium catecholate, implying that selectivity for iron(II) also reflects the cofactor selectivity catechol binds to the iron centre as the monoanion,64 the found in Nature. However, the factors that control the same conclusion being reached from EXAFS studies on the processing of a common hydroperoxide intermediate to 2,3-CTD–catechol complex.65 However, very different intradiol or extradiol products are still not fully understood: product ratios are obtained using monosodium catecholate, we have suggested that stereoelectronic factors are import- as shown in Figure 18. In the absence of pyridine, using ant,50,54 whereas Que and co-workers have suggested that monosodium catecholate, a 1:15 ratio of extradiol to the co-ordination geometry around the iron centre is intradiol products is formed, implying that pyridine has a important.62

Figure 18. TACN model reaction. 7086 T. D. H. Bugg / Tetrahedron 59 (2003) 7075–7101 3. Arene (dihydroxylating) dioxygenases

The initial step in the bacterial degradation of arene hydrocarbons is usually a cis-dihydroxylation of the aromatic ring, to give a cis-dihydro diol. This reaction is catalysed by a family of non-heme iron-dependent dioxy- genases, of which the best studied is naphthalene dioxy- genase (Fig. 19).

Figure 19. Electron transport chain in naphthalene 1,2-dioxygenase.

3.1. Napthalene dioxygenase: structure and catalytic mechanism Figure 20. Active site of naphthalene 1,2-dioxygenase from Pseudomonas sp., complexed with indole and dioxygen. The enzyme consists of three components, which form an electron transfer chain: an NADH-dependent flavoprotein reductase;68 a ferredoxin containing two [2Fe2S] Rieske dioxygenase have both been shown to possess mono- iron–sulfur clusters;69 and a Rieske oxygenase containing oxygenase activity using alternate substrates, suggesting both a [2Fe2S] Rieske iron–sulfur cluster and a mono- that dihydroxylation is not a concerted process.76,77 By nuclear iron(II) centre in the enzyme active site.70,71 analogy with heme and non-heme mono-oxygenase enzymes, this type of reactivity suggests the possible The crystal structure of the terminal dioxygenase com- involvement of iron–oxo intermediates. It has also been ponent of naphthalene dioxygenase was solved by Kauppi observed that processing of benzene by naphthalene et al. in 1998, revealing a mononuclear iron(II) centre in the dioxygenase leads to the production of hydrogen peroxide, active site, co-ordinated by His-208, His-213, and a via uncoupling of oxygen activation from substrate bidentate Asp-362.72 The structure revealed that the hydroxylation.78 These observations suggest that dioxygen mononuclear iron(II) centre was positioned within 12 A˚ of is activated via superoxide, which can be further reduced to the [2Fe2S] cluster of another subunit in the a3b3 oxygenase peroxide. domain.72 Refinement of the structure revealed electron density for an indole hydroperoxide, ligated to the iron(II) Single turnover studies of the naphthalene dioxygenase centre, with the indole ring positioned at about 4 A˚ from the reaction have shown that turnover requires reduced enzyme iron(II) centre.73 The presence of indole was believed to and bound substrate, and that one mononuclear iron(II) and arise from the presence of L-tryptophan in the growth one Rieske [2Fe2S] cluster are oxidised during turnover, media, and the observation of a hydroperoxide species resulting in iron(III) at the end of the catalytic cycle.79 suggested that an alkyl hydroperoxide intermediate might Recent Q-band ENDOR studies of naphthalene dioxygenase be formed in the catalytic mechanism.73 Further recent using 2H-naphthalene have revealed that the distance from crystallographic studies have yielded the structures of the iron(II) centre to the substrate C–D shifts from 4.4 to ternary complexes with substrates (naphthalene or indole) 5.0 A˚ upon reduction of the [2Fe2S] cluster, suggesting a and dioxygen.74 Remarkably, dioxygen was found to be conformational change that may be significant during the bound side-on to the iron(II) centre, with Fe–O distances of catalytic cycle.80 1.8 and 2.0 A˚ , and an O–O distance of 1.4 A˚ . The aryl substrate was positioned slightly further from the iron(II) Although there is at present no clear consensus on a centre, but close to the bound oxygen (Fig. 20).74 mechanism for cis-dihydroxylation, the observations that dihydroxylation of the substrate and reduction of dioxygen Despite the detailed structural data for this enzyme, there can be uncoupled under certain conditions argue in favour are relatively few insights into the catalytic mechanism for of a non-concerted, stepwise mechanism for catalysis. One this remarkable transformation. The original mechanistic possible mechanism (Fig. 21(A)) would be activation of proposal for dihydroxylation, by Gibson in 1970, involved dioxygen via reduction to superoxide, followed by reaction the formation of a dioxetane intermediate, followed by two- with the aryl substrate to generate a substrate hydroper- electron reduction by the enzyme.75 This mechanism now oxide, which would formally be a radical intermediate, but seems unlikely, given the presence of iron–sulfur clusters in which might complex to the iron centre to form an the enzyme, suggesting one-electron transfers in the organometallic species. O–O bond cleavage would give catalytic mechanism. Toluene dioxygenase and naphthalene an iron(V)–oxo species which could effect the second T. D. H. Bugg / Tetrahedron 59 (2003) 7075–7101 7087

Figure 21. Possible stepwise (A) and concerted (B) mechanisms for arene dioxygenases. hydroxylation on the same face of the substrate. Alterna- Further tetradentate ligands were subsequently found that tively, Wolfe et al. have suggested that O–O bond cleavage could catalyse this reaction: ligand 26 complexed with could occur first, to give an OvFe(V)–OH intermediate iron(II) gave a 75% yield of epoxide product, while the which could effect dihydroxylation (Fig. 21(B)) in a similar chiral ligand 27 gave an enantiomeric excess of 82% in the 85 fashion to the dihydroxylation of alkenes by NaIO4 or cis-diol product formed from trans-2-octene. 79 OsO4.

Attempts to trap a putative substrate radical intermediate using cyclopropyl benzene (24) by Bui et al. resulted in no cyclopropyl ring opening (Fig. 18), however, these studies do not rule out a fleeting radical intermediate.81 Recent 2H- labelling studies on the hydroxylation of indene by NDO are consistent with a substrate radical intermediate which can rotate prior to hydroxylation by an active oxygen species.82 The application of the arene dioxygenases for the prep- aration of a wide range of optically active cis-dihydro-diols has been recently reviewed.83

3.2. Model reactions for non-heme iron-catalysed cis- dihydroxylation

Although the dihydroxylation of alkenes by OsO4 and RuO4 is well known, there were until recently no examples of non- heme iron model complexes that could effect this trans- formation, and hence mimic the arene dioxygenases. In A study of a series of ligands of this type showed that two 1999, Chen and Que reported that an iron (II) complex of types of behaviour were observed.86 Ligands such as 26, 6-trimethyl-TPA (25) could catalyse the cis-dihydroxyl- bearing no more than one 6-substituent, formed low-spin 84 87 ation of cyclooctene, using H2O2 as oxidant (Fig. 22). In Fe(III)–OOH intermediates observed previously, and the presence of ten equivalents of H2O2, 4.9 mol equiv. of yielded predominantly epoxide products. Both epoxide cis-diol product were formed. Isotope labelling studies and cis-diol products in these cases incorporated 18O from 18 18 18 v using H2 O2 gave 95% incorporation of two atoms of O, H2 O, consistent with an exchangeable HO–Fe(V) O and 4% incorporation of only one atom of 18O, establishing intermediate. Ligands such as 25, bearing two or more 6- that this is a dihydroxylation process.84 substituents, formed high-spin Fe(III)–OOH intermediates, and yielded predominantly cis-diol products, which incor- 18 18 86 porated two atoms of O from H2 O2. Thus, the key features required for dihydroxylation are: two adjacent co- ordination sites; a high-spin iron centre; and a sterically demanding co-ordination sphere. These factors appear to promote O–O cleavage, as shown in Figure 23.

Manganese(II) catalysts, which are well known to catalyse epoxidation in the presence of H2O2, have also been reported to catalyse cis-diol formation. Attachment of ligand 28 to a silica surface, in the presence of Mn2þ, Figure 22. Conversion by 25 to give cis-diol and epoxide. gave a heterogeneous catalyst with enhanced ability to 7088 T. D. H. Bugg / Tetrahedron 59 (2003) 7075–7101

Figure 23. Proposed catalytic cycle for a model dihydroxylation reaction. catalyse cis-diol formation (37% yield), compared to the A sizeable family of these enzymes are now known, and homogeneous catalyst (,1% yield).88 As yet there are no their enzymology and structures have been recently reports of cis-diol formation from arene substrates. reviewed.91 –93 A number of crystal structures have been obtained for these enzymes, and in each case the mononuclear iron(II) centre is co-ordinated by a His, His, Glu motif observed in other non-heme iron-dependent enzymes (Fig. 25).92,93 Structural studies on clavaminic acid synthase have indicated the structural basis for the separate hydroxylation and oxidative cyclisation/desaturation reac- tions catalysed by this enzyme.94

4. a-Ketoglutarate-dependent dioxygenases

4.1. Enzymology of a-ketoglutarate-dependent dioxygenases

A further class of non-heme iron-dependent dioxygenases catalyse monohydroxylation reactions, and desaturation reactions, of a variety of organic compounds, utilising the co-substrate a-ketoglutarate, which is converted during the reaction to succinate. Prolyl hydroxylase was the first a- ketoglutarate-dependent dioxygenase to be identified in 1967 by Udenfriend.89 This enzyme catalyses the hydroxy- lation of prolyl residues in collagen to 4-hydroxy-prolyl residues (Fig. 24). One oxygen atom is incorporated from dioxygen into the hydroxylated product, and one into succinate.90

Figure 25. Active site of clavaminic acid synthase. By analogy with cytochrome P450-catalysed hydroxylation reactions, it has been proposed that the catalytic mechanism of these enzymes involves a high-valent iron–oxo inter- mediate.95,96 The oxygen atom of this intermediate has been shown to undergo partial exchange with the oxygen atom of water in deacetoxy/deacetylcephalosporin C synthase,97 p-hydrophenylpyruvate hydroxylase (which utilises an a-ketoacid group in the substrate for oxidative decarboxyl- Figure 24. Prolyl hydroxylase. ation, rather than an a-ketoglutarate co-substrate),98 T. D. H. Bugg / Tetrahedron 59 (2003) 7075–7101 7089 a-ketoisocaproate oxygenase,99 and lysyl hydroxylase,100 intermediate. The iron–oxo species then effects hydroxyl- but not in prolyl hydroxylase.101 Evidence for a radical ation of the substrate, probably via hydrogen atom intermediate has been provided by the mechanism-based abstraction to form a substrate radical intermediate inactivation of prolyl hydroxylase by a substrate analogue (Fig. 26). (29) containing a labile N–O bond adjacent to the site of hydroxylation.102 The diverse chemistry that can be carried out by the putative iron(IV)–oxo intermediate is elegantly illustrated by the recent discovery of a new member of this family of enzymes, which catalyses the conversion of p-hydroxyphe- nylpyruvic acid into p-hydroxyphenyl-lactic acid, as part of the biosynthetic pathway to the glycopeptide antibiotic vancomycin.103,104 This enzyme shares 34% amino acid sequence identity with p-hydroxyphenylpyruvate dioxygen- ase, which converts the same substrate into homogentisic acid, as part of the tyrosine degradative pathway.105 Both Therefore, the catalytic mechanism is believed to enzymes effect the oxidative decarboxylation of p-hydro- proceed via formation of an iron(III)–superoxide complex, phenylpyruvate to p-hydroxyphenylacetate, generating the followed by attack of superoxide upon the ketone carbonyl iron(IV)–oxo intermediate, which then either carries out group of a-ketoglutarate. Decarboxylation of the resulting hydroxylation in the benzylic position, or electrophilic hydroperoxide intermediate, with cleavage of the O–O hydroxylation at C-1 of the aromatic ring, followed by a 1,2- bond, then generates succinate and an iron(IV)–oxo alkyl shift, as shown in Figure 27.

Figure 26. Catalytic mechanism for a-ketoglutarate-dependent dioxygenases.

Figure 27. Dioxygenase-catalysed conversion of p-hydroxyphenylpyruvic acid into p-hydroxyphenyllactate or homogenisic acid. 7090 T. D. H. Bugg / Tetrahedron 59 (2003) 7075–7101 conversion of polyunsaturated fatty acids into lipid hydro- peroxides. In plants, the common substrate for lipoxygenase is linoleic acid (32). In mammals, the most common substrate is arachadonic acid (33), which is converted via its hydroperoxide into the leukotriene family of physiological regulators. A related process, which will be considered in Section 5.2, is the conversion in mammals of arachadonic acid (33) and dioxygen into prostaglandin G2 (34), catalysed by cyclo-oxygenase, which does not require an iron cofactor (Fig. 29). Given the physiological importance of the products of these enzymes, there is a great deal of interest

Figure 28. Reaction of model complex 31. 4.2. Model chemistry for a-ketoglutarate-dependent in the inhibition of these enzymes, the latter being the hydroxylation molecular target for aspirin.

The first reported iron(II)-containing model for this class of 5.1. Lipoxygenases enzyme was an iron(II)–TLA complex (30), which upon exposure to oxygen effected the oxidative decarboxylation The lipoxygenases contain a single mononuclear iron of a benzoylformate ligand to benzoic acid in 98% yield.106 centre, which in its resting state is in the þ2 oxidation In 1999 Hegg et al. reported that exposure of mononuclear iron(II) complex (31) to oxygen led to the concomitant oxidative decarboxylation of a benzoylformate ligand, and the hydroxylation of a nearby aryl ring in the ligand (Fig. 28).107 The reactivity of this complex therefore effectively mimics the reactivity of this class of dioxygenase.

Resonance Raman spectroscopic studies of the ligand 31 and related ligands complexed with iron(II) and a-ketoacid have revealed peaks at 460–490 and 1630–1690 nm due to chelation of the a-ketoacid group with iron(II).108 Shifts of these peaks upon substrate binding in oxygenase TauD upon substrate binding imply a change from 6- to 5-co-ordinate iron(II), thus providing a vacant site for dioxygen binding.108 Studies of oxygenase TfdA, which catalyses the oxidative dealkylation of the herbicide 2,4-dichlorophe- noxyacetic acid (2,4-D), have shown that this type of self- hydroxylation also occurs in the active site of the protein.109 Incubation of apo-enzyme with Fe2þ and a-ketoglutarate gives a pink chromophore (lmax 530 nm). Upon addition of 2,4-D and exposure to dioxygen, a new blue chromophore (lmax 580 nm) is produced, which is shown to contain a 5- hydroxytryptophan residue, formed by self-hydroxylation of Trp-112.109

5. Lipoxygenase and cyclo-oxygenase

The lipoxygenases are a family of non-heme iron-dependent dioxygenases, found in animals and plants, that catalyse the Figure 29. Reactions catalysed by lipoxygenase and cyclo-oxygenase. T. D. H. Bugg / Tetrahedron 59 (2003) 7075–7101 7091 state. The crystal structures and plant and mammalian intermediate, and subsequent reaction with dioxygen.123 In lipoxygenases show that the iron(II) centre is ligated by 1988, a tyrosyl protein radical was identified in the active three histidine residues, the amide side-chain of an site of PGHS, generated by the peroxidase activity of the asparagine residue, and the carboxylate of the C-terminal heme cofactor, and this protein radical is believed to initiate amino acid.110 – 112 In order to be catalytically active, the a radical-based mechanism.124 Abstraction of the C-13 proS iron(II) centre is oxidised to a yellow iron(III)–hydroxide hydrogen generates a substrate radical, which has been species, for which the oxidant appears to be the product detected by EPR spectroscopy,125 and has recently been hydroperoxide.113,114 Thereafter, the reaction is believed to characterised as a pentadienyl radical using deuterium- proceed via activation of the substrate, prior to reaction with labelled substrates.126,127 The co-crystallisation of dioxygen. There is evidence to support the formation of a arachadonic acid at the active site of cyclo-oxygenase has substrate radical intermediate, which can be generated from revealed the conformation of the bound substrate, which the iron(III) centre and linoleic acid in the absence of rationalises the stereochemistry of the enzyme-catalysed oxygen.115,116 An alternative organoiron intermediate has process, as shown in Figure 31.128,129 In common with also been proposed.117 Reaction of the activated substrate lipoxygenase, the mechanism involves activation of the with dioxygen then gives a hydroperoxy radical, which substrate as a radical intermediate, followed by reaction is reduced by iron(II) to give the product hydroperoxide with dioxygen, but the subsequent cyclisation is unique to (Fig. 30). this enzyme.

Hydrogen atom abstraction is the rate determining step for 5.3. Model chemistry for hydrogen atom abstraction these enzymes, which exhibit extremely high kinetic isotope 118 –120 effects on kcat (kH/kD¼30280). Hydrogen tunnelling The key initial step in the lipoxygenase and cyclo- has been invoked to rationalise the magnitude of these oxygenase reactions is C–H cleavage via abstraction of a effects, which are temperature-independent, and occur in hydrogen atom. There are only a small number of metal- several site-directed mutants of soybean lipoxygenase-1.121 based systems able to effect this reaction. Mayer and co-workers have shown that chromyl chloride and per- 5.2. Cyclo-oxygenase manganate metal-oxo species can carry out hydrogen atom abstraction,130 and more recently have discovered an Prostaglandin H synthase (PGHS), or cyclo-oxygenase, is a iron(III) tris(2,20-biimidazoline) catalyst for this step.131 heme-containing protein that catalyses the first committed step in the biosynthesis of the prostaglandin and thrombox- Pauly et al. have reported a closer mimic of the ane family of physiological effector molecules in mammals, lipoxygenase active site, namely the penta-coordinate namely the conversion of arachadonic acid (33)into iron(III) complex 35, which is able to oxidise 1,4- prostaglandin G2 (34). Since the products are important cyclohexadiene, via hydrogen atom abstraction, to ben- mediators in inflammation, there is considerable interest in zene.132 A crystal structure of the iron(III) complex showed this enzyme for the development of anti-inflammatory a short Fe–O bond length of 1.78 A˚ to the bound methanol agents.122 ligand, similar to the Fe–O distance of 1.88 A˚ found in the lipoxygenase iron(III) hydroxide structure. A kinetic isotope Upon discovery of this pathway by Samuelsson in 1967, he effect of 2.7 was measured for this reaction, indicating that proposed a mechanism involving the formation of a radical C–H cleavage is partially rate limiting.132

Figure 30. Catalytic mechanism for lipoxygenase. 7092 T. D. H. Bugg / Tetrahedron 59 (2003) 7075–7101

Figure 31. Catalytic mechanism for cyclo-oxygenase.

An unusual feature of IDO is that it can be activated by the binding of superoxide to the ferric enzyme, as well as by the binding of dioxygen to the ferrous enzyme;133 however, the high levels of superoxide dismutase activity in vivo cast doubt on the physiological role of this activity.2 The catalytic mechanism for IDO and TDO is believed to proceed via the formation of a hydroperoxide at C-3 of the indole ring, followed either by dioxetane formation or Criegee rearrangement.134 Formation of the hydroperoxide could either take place via nucleophilic attack upon heme-bound dioxygen,134 or via the for- mation of an indole radical, followed by recombination 6. Other metal-dependent dioxygenases with iron(III)–superoxide.135 Fluorinated tryptophan ana- logues show reduced vmax,andinthecaseof7-fluoro-Trp Several other metal-dependent dioxygenases have been do not act as a substrate for IDO, implying a need for an identified, which are outside the classes discussed above. electron-rich aryl ring.2,135 Possible catalytic mechanisms The first three catalyse the oxidative cleavage of an alkene are illustrated in Figure 33. CvC bond; the next two catalyse oxidative cleavage of a C–C single bond; the final example catalyses the oxidation Indoleamine 2,3-dioxygenase has recently sprung to of a thioether to a sulfone. prominence in cell biology, with the discovery that the expression of IDO activity in the mouse foetus repressed the 6.1. Indoleamine 2,3-dioxygenase and tryptophan 2,3- maternal T-cell activity and hence protected the foetus from dioxygenase the maternal immune system.136 Pregnant mice treated with the IDO inhibitor 1-methyltryptophan rejected the embryos L-tryptophan is degraded via oxidative cleavage of the C-2, via their immune system, thus either IDO itself or a product C-3 bond, to give N-formyl-kynurenine. Tryptophan 2,3- of tryptophan catabolism is able to suppress the maternal T- dioxygenase (TDO), found in mammals and bacteria, is cell activity.136 In addition, IDO is expressed in response to selective for cleavage of tryptophan, whereas indoleamine interferon g from activating T-cells, inhibiting T-cell 2,3-dioxygenase (IDO), found only in mammals, is able to proliferation137 and contributing towards the antiviral cleave other indoleamine derivatives such as tryptamine and activity of interferon g.138 IDO expression in macrophages serotonin (Fig. 32). Both enzymes are heme-containing also leads to the production of the neurotoxin quinolinic proteins, unlike the catechol dioxygenases involved in other acid,139 and thus IDO appears to be implicated in several aromatic degradation pathways.2 mammalian regulatory pathways.

Figure 32. Reaction catalysed by indoleamine 2,3-dioxygenase. T. D. H. Bugg / Tetrahedron 59 (2003) 7075–7101 7093

Figure 33. Possible catalytic mechanisms for indoleamine 2,3-dioxygenase.

6.2. Quercetin 2,3-dioxygenase in a bent conformation at this point, thereby lowering the activation energy by relief of strain.142 As shown in Figure Flavonoids are commonly found in plant material, and can 34, the catalytic mechanism then proceeds via intramole- be degraded by Aspergillus using the enzyme quercetin 2,3- cular nucleophilic attack of a hydroperoxide anion, followed dioxygenase. This enzyme is unique in that it is the only by cheletropic ring opening, releasing CO. known copper-dependent dioxygenase enzyme.140,141 The reaction is also unusual in that oxidative cleavage of two Studies of the kinetics and mechanism of the base-catalysed carbon–carbon bonds takes place, liberating carbon mon- oxygenation of flavonols in DMSO/water show general base oxide as a by-product. The structure of Aspergillus catalysis, and are consistent with the reaction of a stabilised japonicus quercetin 2,3-dioxygenase, solved in 2002 by carbanion with dioxygen, with no radical intermediates Steiner et al. revealed that the mononuclear copper(II) being detected.143 Although it is possible that the copper centre is positioned close to the C-3 hydroxyl group, centre is involved in oxygen activation (i.e. reaction with a allowing radical formation at this centre.142 Reaction with copper(I) intermediate), modelling of putative intermediates dioxygen via C-2 is assisted by the binding of the substrate suggests that this is unlikely.142 The role of the copper

Figure 34. Reaction mechanism for quercetin 2,3-dioxygenase. 7094 T. D. H. Bugg / Tetrahedron 59 (2003) 7075–7101 centre in the enzyme therefore appears to be to activate the porphyrin ring, which bind the ends of the substrate. substrate for reaction with dioxygen. Incubation with b-carotene and t-butyl hydroperoxide yields retinal as the major reaction product.150 6.3. b-Carotene dioxygenase

The biological source of retinal, used as a cofactor in the light-sensing protein rhodopsin, is well known to proceed via oxidative cleavage of b-carotene, as shown in Figure 35 (hence a deficiency in vitamin A causes night blindness).144 Until recently, there has been little information regarding the dioxygenase enzyme that catalyses this reaction. However, the genes encoding this enzyme from chicken intestinal mucosa,145 Drosophila melanogaster,146 and mouse kidney147 have been cloned and expressed, and a series of mechanistic studies carried out on the recombinant enzyme.148 Several modified substrate analogues were synthesised and tested, revealing that the enzyme is 6.4. Methionine salvage pathway dioxygenases selective for a rod-like polyene substrate, but that some modifications to the substituents of the polyene are tolerated The biological cofactor S-adenosyl methionine can be by the enzyme.149 converted metabolically into 50-thiomethyladenosine, which is recycled via opening of the ribose ring to the amino acid L-methionine. An unusual transformation in this pathway is the oxidative cleavage of aci-reductone (37)to2- keto-4-thiomethylbutyrate and formic acid, while a related transformation converts the same substrate to 3-thiomethyl- propionate, formic acid, and CO (Fig. 36).151,152

Abeles and co-workers have shown that these transform- ations are catalysed in Klebsiella pneumoniae by two closely related dioxygenase enzymes, which incorporate 18 18 two atoms of Tryptophan 2,3-dioxygenase O from O2 into their respective products.153 Remarkably, the two dioxygenases share the same polypeptide sequence, but contain different metal ion cofactors.154 Dioxygenase ARD Figure 35. Reaction catalysed by b-carotene dioxygenase. (which catalyses two C–C cleavages) contains a single iron(II) cofactor, whereas dioxygenase ARD0 (which catalyses one C–C cleavage) contains a single nickel(II) 17 18 154 Incubations carried out in O2 and H2 Ousingan cofactor. The two modes of oxidative cleavage can be asymmetric substrate revealed approximately 50 atom% explained by the formation of a common hydroperoxide incorporation of oxygen into each aldehyde product from intermediate at C-1, followed either (in ARD0)by each source.148 One possible interpretation is that the nucleophilic attack upon C-2 to form a dioxetane, or (in enzyme converts the central alkene to an epoxide, which is ARD) by nucleophilic attack upon C-3 to form a five- attacked by water to give a diol, which is then oxidatively membered endo-peroxide (Fig. 37).155 A cyclopropyl cleaved.148 Analysis of the purified enzyme using plasma substrate analogue (38) inactivates both dioxygenases, emission analysis has shown that the holoenzyme contains providing some evidence for a radical-mediated formation one equivalent of iron. of the hydroperoxide.155 The two oxidative cleavages can be observed non-enzymatically, via base-catalysed auto-oxi- Woggon and co-workers have devised a model catalyst for dation.155 The choice of metal ion cofactor appears to b-carotene cleavage, involving a Ru porphyrin (36), to dictate the choice of reactivity of the hydroperoxide which two cyclodextrins are attached at the periphery of the intermediate.

Figure 36. Reactions catalysed by dioxygenases ARD and ARD0. T. D. H. Bugg / Tetrahedron 59 (2003) 7075–7101 7095 via hydrolytic C–C cleavage, however, Straganz et al. have discovered an unusual oxidative cleavage of acetylacetone in Acinetobacter johnsonii (Fig. 38).157 The purified enzyme contains 0.4–0.8 mol Fe2þ and 1.2–2.0 mol Zn2þ per mol protein, and thus the enzyme appears to require both iron(II) and zinc(II).157 A plausible mechanism would involve the formation of a C-3 hydroperoxide, followed by either dioxetane formation or Criegee rearrangement.

Figure 38. Reaction catalysed by acetylacetone dioxygenase.

6.6. Cysteine dioxygenase

Cysteine is metabolised in mammals via oxidation to Figure 37. Catalytic mechanisms for dioxygenases ARD and ARD0. cysteinesulfinic acid and taurine (Fig. 39), which is conjugated with cholic acid to make the bile salt taurocho- line. Rat liver cysteine dioxygenase has been purified, and the purified enzyme contains 0.8–0.9 mol iron per enzyme subunit.158 The enzyme has been shown to incorporate two 18 18 atoms of Tryptophan 2,3-dioxygenase O from O2, with- out exchange with water.159 The cDNA encoding this enzyme has been cloned.160

The structure of nickel(II)-containing ARD has been solved by NMR spectroscopy.156 The mononuclear nickel(II) centre has an octahedral geometry, and is ligated by four 7. Cofactor-independent dioxygenases protein ligands: His-96, His-98, Glu-102, and His-140. The substrate is believed to co-ordinate to the two vacant co- Although the great majority of dioxygenases utilise a metal ordination sites as the dianion. A structural similarity is cofactor to assist oxygen activation, a small number of observed with enzymes of the cupin superfamily.156 enzymes have been isolated that require neither metal ion nor organic cofactor. In these cases, reaction of a substrate 6.5. Acetylacetone dioxygenase carbanion with dioxygen can be rationalised mechanisti- cally, if a stable substrate radical can be accessed, as shown in Bacterial degradation of 1,3-diketones usually takes place Figure 40.

Figure 39. Reaction catalysed by cysteine dioxygenase.

Figure 40. General scheme for the reaction of carbanions with dioxygen.

Figure 41. Biosynthesis of the antibiotic LL-C10037a (41). 7096 T. D. H. Bugg / Tetrahedron 59 (2003) 7075–7101

Figure 42. Catalytic mechanism for epoxyquinone-forming dioxygenase.

7.1. Epoxyquinone natural product biosynthesis in oxoquinoline 2,4-dioxygenase) and HOD (1H-3-hydroxy-4- Streptomyces oxoquinaldine 2,4-dioxygenase) have been purified, and neither contains a metal ion nor an organic cofactor.168 Studies of the biosynthesis of the antibiotic LL-C10037a (41)inStreptomyces LL-C10037 have led to the identifi- Amino acid sequence alignments have revealed, remark- cation of a novel dioxygenase enzyme activity which ably, that QDO and HOD are members of the ab-hydrolase catalyses the conversion of 2,5-dihydroxyacetanilide (39)to superfamily, containing a Ser-His-Asp triad, the normal the epoxyquinone 40 (Fig. 41). This dioxygenase has been function of which is to participate as a nucleophile in amide purified, and the purified enzyme requires neither metal ion and ester hydrolysis reactions.169 Replacement of Ser-95 in nor organic redox cofactor.161 A second dioxygenase was QDO and Ser-101 in HOD by Ala by site-directed also identified that converts 39 to the opposite enantiomer of mutagenesis gave mutant enzymes with approximately 40, also without a cofactor requirement.161 10% wild-type activity, indicating that the putative active site serine is not essential for activity.170 The probable Similar transformations of p-hydroquinone substrates into epoxyquinones have been reported in Penicillium patu- lum,162 andinthemammalianvitaminK-dependent carboxylase.163,164 Isotope labelling studies have estab- 18 18 lished that one atom of O is incorporated from O2 into the epoxide oxygen, and approximately 0.20 atom 18O into the C-4 ketone of 40 (the remainder being exchanged with solvent water), thus verifying that the enzyme is a dioxygenase.165 The proposed mechanism (Fig. 42) involves reaction of the substrate with dioxygen at C-4 to form a hydroperoxide, which undergoes nucleophilic attack on the neighbouring alkene to form a dioxetane, which is opened to form the epoxide.161,165

The biosynthesis of tetracenomycin C (42)inStreptomyces glaucescens (Fig. 43) proceeds via a triple hydroxylation reaction, in which two oxygens are derived from dioxygen, and one oxygen from water.166 This labelling pattern can be explained by a similar transformation to give an epoxyqui- none intermediate, followed by base-catalysed epoxide opening by water, and ketone reduction.166

7.2. Oxidative cleavage of 3-hydroxy-4-oxoquinolines by bacterial 2,4-dioxygenases

The bacterial degradation of quinoline heterocycles pro- ceeds via oxidation to 3-hydroxy-4-oxoquinolines, which are substrates for a novel family of dioxygenases.167 The reaction is similar to the copper-dependent quercetin dioxygenase (Section 6.2), involving oxidative cleavage of two C–C bonds, and liberation of carbon monoxide, however, unlike QDO, these enzymes have no cofactor requirement. Two dioxygenases QDO (1H-3-hydroxy-4- Figure 43. Biosynthesis of tetracenomycin C (42). T. D. H. Bugg / Tetrahedron 59 (2003) 7075–7101 7097 eliminate water to give a ketone product, or be reduced to give an alcohol product.172

In conclusion, dioxygenase enzymes catalyse a diverse range of remarkable biochemical reactions that at first glance appear to defy chemical logic and lack chemical precedent. However, detailed mechanistic and structural studies have elucidated the essential features of many of these transformations, and in many cases our understanding of these reactions has been confirmed by the design of biomimetic model reactions. Although we can rationalise many transformations mechanistically, the exquisite selec- tivity of the enzyme-catalysed reactions is, nevertheless, remarkable.

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Biographical sketch

Tim Bugg (born 1965) is Professor of Biological Chemistry at the University of Warwick. Following his PhD studies with Dr C. Abell at the University of Cambridge, he spent two years as a SERC/NATO postdoctoral research fellow in the laboratory of Professor C. T. Walsh at Harvard Medical School. In 1991 he began his academic career as a lecturer in organic chemistry at the University of Southampton, before moving to Warwick in 1999. His research interests are in the study of enzyme mechanisms, principally enzymes involved in the bacterial degradation of aromatic compounds, and enzymes involved in bacterial peptidoglycan biosynthesis. He enjoys playing and coaching volleyball, cycling, and playing the violin.