Non-Heme Mono-Iron Enzymes: Co-Substrate-Independent Dioxygen Activation

Yisong Guo,1* Wei-chen Chang,2* Jikun Li,1 and Madison Davidson 2

1 Department of Chemistry, Carnegie Mellon University, Pittsburgh, PA 15213 2 Department of Chemistry, North Carolina State University, Raleigh, NC 27695

1. Introduction ...... 2 2. Enzymes that Act on Organophosphonic Acids ...... 2 2.1 Introduction ...... 2 2.2 Structural Considerations ...... 3 2.3 General Reaction Mechanisms of HEPD and MPnS ...... 3 2.4 General Reaction Mechanism of HppE ...... 4 3. Thiol Oxidation Enzymes ...... 6 3.1 Introduction ...... 6 3.2 Isopenicillin N Synthase ...... 6 3.3 Cysteine Dioxygenase ...... 9 3.4 Sulfoxide Synthases, EgtB and OvoA ...... 13 4. Ring-Cleaving Dioxygenases ...... 16 4.1 Introduction ...... 16 4.2 Structural Considerations ...... 17 4.3 General Reaction Mechanism of Extradiol-Cleaving Dioxygenases...... 19 4.4 General Reaction Mechanism of Intradiol-Cleaving Dioxygenases...... 23 4.5 Other Ring-Cleaving Dioxygenases...... 25 5. Carotenoid Cleavage Oxygenases ...... 29 5.1 Introduction ...... 29 5.2 Structural Considerations ...... 29 5.3 General Mechanism ...... 31 6. Lipoxygenases ...... 32 6.1 Introduction ...... 32 6.2 Structural Considerations ...... 33 6.3 General Mechanism ...... 34 7. Rieske Oxygenases ...... 35 7.1 Introduction ...... 35 7.2 Structural Considerations ...... 37 7.3 General Mechanism ...... 38 8. Concluding Remarks ...... 40 9. Acknowledgements ...... 41 10. References ...... 41

1 1. Introduction

Non-heme mono-iron (NHMI) dioxygen (O2) activating enzymes are widely distributed in nature. They are involved in many fundamental biological pathways, including DNA repair, gene regulation, O2 sensing, natural product biosynthesis, and organic molecule metabolism. By using O2 as the terminal oxidant, NHMI enzymes catalyze a stunningly broad array of oxidative reactions, including some of the most challenging chemical transformations, such as hydroxylation and halogenation on unactivated carbon-hydrogen bonds and carbon-carbon (C(sp2)-C(sp3)) bond formation. Two general strategies are utilized by NHMI enzymes to activate O2. In one strategy, O2 activation by NHMI enzymes is coupled with the 2-electron oxidation of co-substrate (such as α-ketoglutarate, αKG, or pterin) in order to achieve 4-electron reduction of O2 and generate key reactive intermediates to carry out the 2-electron oxidation of the target substrate. In the other strategy, co-substrates are not required; rather all 4 electrons needed to reduce O2 are provided by the substrates in many cases. For co-substrate-depedent enzymes, ferryl or Fe(IV)=O intermediates are usually observed or proposed to be the key reactive intermediate. For co-substrate-independent enzymes, different types of reactive intermediates including ferryl intermediates could be involved in enzyme catalysis. Although different O2 activation strategies are used, the coordination environments of the mono-iron centers in NHMI enzymes are quite similar and consist at least two histidine residues and one or two other amino acid residues, such as aspartate, glutamate, histidine, or tyrosine. The preceding chapter has focused on the mechanistic understanding on the co- substrate-dependent NHMI enzymes, such as αKG-dependent (Fe/αKG) or pterin-dependent NHMI enzymes. Herein, we provide a detailed summary of studies on the co-substrate- independent NHMI enzymes with emphases on the iron coordination environments of various enzymes and the reaction mechanisms they support. Readers are also encouraged to refer to many excellent review articles and book chapters published in recent years on this subject.1,2,11–20,3,21– 23,4–10

2. Enzymes that Act on Organophosphonic Acids

2.1 Introduction

Organophosphonic acid containing natural products are widely distributed bioactive secondary metabolites. Some of the enzymatic transformations involved in making these molecules are chemically unprecedented and have caught the attention of (bio)chemists. Efforts have been put forward to elucidate the plausible reaction pathways for these unique transformations. Herein, we will discuss the recent developments in our understanding of three co-substrate-independent NHMI enzymes, (S)-2-hydroxypropylphosphonic acid epoxidase (HppE), 2- hydroxyethylphosphonate dioxygenase (HEPD) and methylphosphonate synthase (MPnS), which respectively catalyze C-O bond formation in fosfomyin (2) biosynthesis and C-C bond cleavage in the formation of hydroxymethylphosphonate (HMP, 4) and methylphosphonate (MPn, 5) (Scheme 1).24–26

Scheme 1. HppE-, HEPD- and MPnS-Catalyzed Transformations.

2.2 Structural Considerations

The iron binding motifs in HppE, MpnS, and HEPD all consist of two histidine residues and an additional protein residue as either glutamate (Glu) or glutamine (Gln). In HppE, the ferrous iron in the active site is ligated by a 2-His-1-Glu facial triad. 27 In MPnS, a 2-His-1-Gln facial triad coordinates the iron center.28 In HEPD, both facial triads have been adopted where class I HEPD uses a 2-His-1-Glu combination and class II HEPD utilizes a 2-His-1-Gln triad.25,28 More recently, it has been discovered that class II HEPD can be functionally converted to MPnS by point mutations in the corresponding DNA.28 Even though the chemical outcomes of HppE-, HEPD- and MPnS-catalyzed reactions are distinct, the three enzymes show high sequence identity and all belong to the cupin superfamily where the active site is situated in the β-sheet domain. X-ray structure comparison also reveals similar folds for the protein chains in dimeric HEPD and MPnS and the tetrameric HppE. 2.3 General Reaction Mechanisms of HEPD and MPnS

Insight into the HEPD- and MPnS-catalyzed reaction mechanisms has arisen through the utilization of a combination of mechanistic approaches, namely product characterization, isotope tracking, kinetic isotope studies, mutagenesis, protein structure structural determination, transient kinetic studies and reaction intermediate characterization.25,26,29–35 During HEPD catalysis, the reaction is likely initiated via abstraction of the C2 pro-S hydrogen atom by a ferric-superoxide species. It is followed by an electron transfer step to form the corresponding ferrous hydroperoxide and an aldehyde. Reaction of the ferrous hydroperoxide with the aldehyde produces a bridged peroxo species that can undergo homolytic O-O bond cleavage followed by an β-scission to generate phosphonomethyl radical (MPn●) and a ferric hydroxide intermediate. Formation of the C-O bond between the resulting radical and the Fe(III)-OH species produces hydroxymethylphosphonate and regenerates the Fe(II) (Scheme 2). More recently, the observation of an Fe(IV)-oxo species through transient state kinetics and Mössbauer spectroscopy has revealed an alternative pathway involving this Fe(IV)-oxo intermediate.33 Instead of the bridged peroxo species, the Fe(IV)-oxo species is formed and serves as the key intermediate to carry out O-H bond cleavage (Scheme 2).

3 A similar reaction pathway has been proposed for the MPnS-catalyzed reaction where the resulting phosphonomethyl radical (MPn●) is proposed to abstract the hydrogen atom from the formate and results in methylphosphonate and a formyl radical. The radical is further converted to CO2 and completes the reaction cycle (Scheme 2). The structural information along with amino acid residue substitution reveals that the geometric arrangement of the formate likely controls the fate of the phosphonomethyl radical and determines the reaction outcome.28 During the HEPD reaction, the formate is possibly positioned far away such that the MPn● preferentially reacts with the hydroxyl group of the Fe(III)-OH. In contrast, the formate hydrogen is in close proximity during the MPnS reaction.

Scheme 2. Top: The proposed reaction pathway of HEPD catalyzed HEP C-C bond cleavage reaction to produce HMP and formate. More recently, the modified pathway has been proposed to account for observation of an Fe(IV)-oxo species (dashed box) during the reaction. Bottom: The proposed reaction of MPnS-catalyzed methylphosphnate formation.

2.4 General Reaction Mechanism of HppE

HppE catalyzes epoxide formation in the last step of fosfomyin biosynthesis (Scheme 3). Although HppE shows high amino acid sequence similarity with HEPD and MPnS, the HppE-catalyzed reaction is distinct from those of HEPD and MPnS.24 While the HEPD and MPnS reactions utilize O2 as the sole oxidant and catalyze C-C bond cleavage, HppE likely uses H2O2 (or O2/reduced flavin) as the oxidant and catalyzes C-O bond formation.36 In addition, by changing the hydroxyl group position, HppE has been demonstrated to catalyze carbonyl group formation and phosphono group migration.36,37

During the HppE-catalyzed epoxidation reaction, after substrate binding, the ferrous ion reacts with hydroperoxide to form the corresponding Fe(IV)-oxo intermediate. Subsequently, the Fe(IV)- oxo abstracts the C1-HR hydrogen and generates an Fe(III)-OH species and the corresponding substrate radical. The reaction is likely followed by the electron transfer step to form the Fe(II)

4 species and a carbocation intermediate, which can be stabilized by one of the oxygens of the phosphonate. Forming the C-O bond between the hydroxyl group and the proposed carbocation completes the stereo-specific epoxide formation (Scheme 3). It is worth mentioning that the reactive Fe(IV)-oxo has not been detected in the HppE reaction. Instead of a carbocation intermediate, it is also possible that the C-O bond formation may proceed through a radical-radical recombination pathway.

In addition to epoxide formation, HppE is found to trigger ketone (C=O) formation when the C2- chiral center is inverted from (S) to (R). Mechanistic studies using isotopes along with structural studies and radical clock analogues suggest that inversion of the C2-chiral center changes the binding of the substrate in the active site and the proposed Fe(IV)-oxo now abstracts the C2 38–42 hydrogen atom, and the C2-substrate radical facilitates C=O bond formation (Scheme 3). Interestingly, using substrate analogues with the hydroxyl group at C1, HppE is found to catalyze 37 phosphono group migration or C=O formation depending on the chirality of the C1 center. Mechanistic studies using isotope labels and mechanistic probes, along with crystallography, reveal that carbon-phosphorus bond cleavage is most likely triggered by a carbocation-induced rearrangement (Scheme 3).37,42

Scheme 3. (a) Four reaction outcomes of the HppE-catalyzed reactions. (b) The proposed reaction pathway of the HppE-catalyzed epoxidation. Both cation- or radical-induced C-O bond formation have

5 been suggested to complete the reaction. (c & d) The current working hypotheses for HppE-catalyzed C=O formation (c) and carbon-phosphorus bond migration (d).

3. Thiol Oxidation Enzymes 3.1 Introduction

Sulfur containing biomolecules play critical roles in many biological pathways.43 For example, cysteine is the source of sulfur for the biosynthesis of most thiols and is required in all cells.43,44 Low-molecular weight thiols, such as glutathione, protect cells from oxidative stress by reacting with reactive oxygen species;45–47 Coenzyme A and its phosphopantetheine precursor are key coenzymes in lipid metabolism and essential to all cells.48 Coenzyme M 49–51 and coenzyme B 52,53 are essential coenzymes involved in methanogenesis, a biological process important in the global carbon cycle.54 Penicillin, belonging to the β-lactam antibiotic family, is one of the most important clinically used sulfur-containing antibiotics.55

NHMI O2 activating enzymes have been discovered in the biosynthesis and degradation of sulfur- containing biomolecules. Several well-studied examples are isopenicillin N synthase (IPNS),56 cysteine dioxygenase (CDO),57 and the sulfoxide synthases EgtB 58 and OvoA.59 IPNS catalyzes a novel four-electron oxidation to convert δ-(L-α-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N, which forms the bicyclic ring of β-lactam in a single step.60,61 CDO catalyzes the first committed step in cysteine catabolism in mammals, where it carries out with the help of O2 the 4-electron oxidation of L-cysteine (L-Cys) to cysteine sulfinic acid (CSA)62,63 to generate taurine or be further converted to pyruvate and sulfite.64 The absence or lack of CDO activity has been linked to a number of diseases, including cancer.57 Finally, EgtB and OvoA are involved in the biosynthesis of ergothioneine (ESH) and ovothiol (OSH),58,59 two low-molecular-weight natural organothiols that are involved in relieving oxidative stress in cells.43,65,66 Both enzymes catalyze a unique sulfur-carbon bond formation reaction between a cysteine dipeptide (γ-glutamylcysteine, γ-GC, in the case of ESH), or cysteine (Cys, in the case of OSH) to the aromatic C-H bond from the imidazole moiety of N-α-trimethylated histidine (TMH, in the case of ESH), or of histidine (His, in the case of OSH). Simultaneously, a sulfoxide moiety is also generated to complete an overall 4-electron oxidation reaction.67

3.2 Isopenicillin N Synthase

A crystal structure of IPNS in complex with MnII was solved by Baldwin and coworkers in 1995.61 This structure reveals a DSBH fold (or cupin fold) for IPNS, which is also present in the Fe/αKG enzymes.17 The metal is coordinated by a 2-His-1-Asp binding motif that is also conserved in αKG/Fe enzymes. The crystal structure of MnII-substituted IPNS further reveals a six-coordinate metal center ligated by His214, Asp216, His270, 2 water molecules, and Gln330. Gln330 is not essential for IPNS activity. This is supported by crystal structures of IPNS in complex with FeII and ACV obtained under anaerobic conditions.68,69 In the IPNS-FeII-ACV complex, the substrate ACV occupies the substrate binding pocket, thus displacing Gln330 from the metal center. The ACV is anchored within the active site by ligation of its thiolate to the iron center and through its two carboxylate groups. The direct ligation between FeII and ACV displaces one of the two metal binding water molecules seen in the MnII-IPNS complex, changing the metal coordination

6 geometry from octahedral to square pyramidal. These observations are fully consistent with an earlier biochemical/spectroscopic study by Lipscomb, Münck and coworkers.70 In that study, the authors concluded that the thiolate on ACV most likely ligates to the iron center based on EPR and Mössbauer measurements on the NO-treated IPNS-FeII-ACV complex. The crystal structure of the NO-ligated IPNS-FeII-ACV complex has also been reported.69 It shows that NO coordinates to the iron center at the vacant coordination site (trans to Asp216) in the IPNS-FeII-ACV complex structure, thus suggesting that this site is highly likely to be the O2-binding site (Figure 1). Based on crystal structures, it is proposed that the presence of O2 promotes the Cα-Cβ bond rotation of the valinyl-isopropyl group and directs the valine β-proton towards the key iron-based intermediate (most likely the Fe(IV)=O species) for the reaction.69,71

H2O ACV Fe

S His270

His214 Asp216

H H N L N SH 3 L H COO- O N D O COO- ACV = ((L-aminoadipoyl)-L-cysteinyl-D-valine) (10)

II Fe , O2 Isopenicillin N synthase 2H2O

H H N N 3 S - COO O N O COO- isopenicillin N (11)

Figure 1. Top: the iron center structure of the IPNS-Fe-ACV-NO complex (PDB: 1BLZ); Bottom: the overall reaction catalyzed by IPNS.

A detailed mechanism (Scheme 4) for IPNS catalysis has been proposed on the basis of enzymological,72 crystallographic,61,69,71 and computational modelling studies 73,74 (see Schofield et al 55 for a more detailed review). The binding of ACV to the Fe(II) center via its cysteinyl thiolate creates a five-coordinate iron center. The vacant coordination site trans to Asp216 is II available for O2 binding, which is suggested by the crystal structure of the NO-bound IPNS-Fe -

7 69 ACV complex. An Fe(III)-superoxo species is proposed to form after O2 binding and to then abstract the pro-3S proton of the cysteinyl β-methylene group, leading to a ferric-hydroperoxide (Fe(III)-OOH) intermediate with a substrate methylene radical. An electron transfer from the methylene radical to the iron center affords a ferrous-hydroperoxide (Fe(II)-OOH) intermediate.75 The protonation of the distal oxygen on Fe(II)-OOH moiety by the valine N-H proton leads to formation of a ferryl intermediate. At the same time, the valine nitrogen attacks the cysteinyl β- carbon to complete the formation of a monocyclic β-lactam. Thus different from Fe/αKG enzymes, the generation of the ferryl intermediate is followed by the 2-electron oxidative cyclization of a thiol-containing substrate, instead of the oxidative decarboxylation of αKG. The determination that the β-lactam ring is formed prior to the formation of the C-S bond of the 5-membered thiazolidine ring comes from kinetic isotope effect studies76 and the crystallographic observation of a monocyclic β-lactam in the case of the substrate analogue δ-(L-α-aminoadipoyl)-L-cysteinyl- 71 S-methyl-D-cysteine (ACMC). The ferryl intermediate then abstracts a H-atom from the Cβ-H bond of the valine to generate a substrate radical that then reacts with the cysteine-thiolate to form the thiazolidine ring of isopenicillin N at the valinyl β-carbon. The observation of deuterium kinetic isotope effects on both the cysteinyl β-carbon and the valinyl β-carbon suggests that both C-H activation steps are rate-limiting.77 Based on this observation, Bollinger, Krebs and coworkers experimentally demonstrated the existence of both the reactive Fe(III)-superoxo species and the 78 ferryl intermediate during IPNS catalysis. By reacting IPNS-Fe(II)-ACV complex with O2, a transient intermediate with an absorption feature at 515 nm has been observed by stopped flow absorption (SF-Abs) measurements that accumulates to a maximum at < 0.1 s. The Mössbauer measurements on freeze-quenched samples confirm the nature of this intermediate as the ferryl 57 intermediate with δ = 0.27 mm/s and |ΔEQ| = 0.44 mm/s. The anisotropy of the Fe hyperfine coupling tensor of this ferryl species as revealed by variable field Mössbauer measurements implicates thiolate binding to the iron center, which is further confirmed by computational modeling. The decay rate constant of this ferryl intermediate shows a large H/D KIE (~ 30) upon introduction of AC[d8-V], confirming that the C-H activation site of the ferryl species is at the valine β-carbon. By using A[d2-C]V (cysteinyl β-carbon deuterated) as the substrate, a second intermediate with optical absorption features at ~ 500 and ~630 nm are observed by SF-Abs measurements, prior to the ferryl intermediate. The Mössbauer measurements on freeze-quenched samples confirm the nature of this intermediate as the Fe(III)-superoxo intermediate with δ = 0.53 mm/s and |ΔEQ| = 1.02 mm/s. Computational modeling has further supported this assignment.

Scheme 4. Reaction Mechanism of the IPNS-Catalyzed Reaction. 3.3 Cysteine Dioxygenase

For CDO, a large number of crystal structures has been reported (a total of 65), almost all of which are eukaryotic CDOs from human, mouse and rat, with only one from a bacterium, Ralstonia eutropha (Protein Data Bank, http://www.rscb.org/pdb/). The overall protein structure has a β- barrel motif characteristic of the cupin superfamily,79 which includes many NHMI enzymes, such as Fe/αKG enzymes, IPNS as described above, and several other dioxygenases. For CDOs, the iron-binding site consists of 3 conserved histidine residues, instead of a 2-His-1-Asp/Glu facial triad as seen in Fe/αKG enzymes. The first two crystal structures reported are from two CDOs with identical sequences in the resting state, one from Mus musculus (mouse) and the other from Rattus norvegicus (rat).80,81 The metal center features a catalytically inactive Ni(II) complex with an octahedral geometry containing 3 histidines and 3 water molecules for the mouse CDO,81 and a Fe(II) complex with a pseudo-tetrahedral geometry with 3 histidines and one water molecule for the rat CDO.80 A six-coordinate metal center is consistent with the XAS results.82 These two crystal structures also reveal a conserved cysteine in all eukaryotic CDOs, Cys93, which is involved in a post-translational modification with Tyr157 to form a cysteinyltyrosine linkage near the active site. The exact function of this cross-linkage has not been fully resolved, but several recent studies have provided critical insights and will be discussed later in this section.

The binding mode of L-cysteine to the iron center of CDO was first revealed by a crystal structure (2.7 Å) from human CDO (Figure 2),83 where the cysteine substrate in the active site coordinates with Fe(II) via its amino nitrogen (trans to His140) and thiolate sulfur (trans to His88). An open coordination site (trans to His86) is available to bind another ligand, such as O2. The bidentate cysteine binding mode is further supported by several spectroscopic studies. An EPR study on NO

9 treated Fe(II)-CDO showed a rare S = ½ {FeNO}7 species, which could only be observed when cysteine substrate was introduced. This was attributed to the bidentate thiol/amine coordination of L-cysteine to the iron center based on DFT calculations in the same study.84 The direct sulfur ligation was also revealed by electronic absorption, MCD, and resonance Raman data on both ferrous and ferric cysteine adducts.85 A Mössbauer study by Jameson and coworkers initially demonstrated a dramatic decrease of isomer shift from the Fe(II)-CDO complex from 1.30 mm/s to 0.8 mm/s upon introduction of L-Cys.86 The authors attributed this change to the binding of L- Cys to the iron center. However, this dramatic change was not observed in a later study by the same group. Instead, upon L-Cys binding, the Mössbauer spectrum of Fe(II)-CDO with isomer shift of 1.22 mm/s was changed to two Mössbauer species with isomer shifts of 1.03 mm/s and 1.10 mm/s, respectively.87 Computational investigations included in this study suggested that the two species correspond respectively to a five- and a six-coordinate Fe(II) center with L-Cys binding. The bidentate Cys binding mode was further confirmed in a crystal structure derived from a bacterial CDO.88 In a crystal structure from rat CDO published by Karplus and coworkers, an iron bound cysteine persulfenate was identified (Figure 2).89 The cysteine-derived portion of the persulfenate is bound to the iron in a bidentate fashion, while the dioxygen-derived portion of the persulfenate is bound to the iron via only one oxygen atom in an end-on fashion. The catalytic relevance of the persulfenate is still unknown. Computational studies do not support the involvement of such a species in the catalytic mechanism based on energetic reason.90,91 However, Karplus and coworkers demonstrated later that this cysteine persulfenate species was observed in multiple high-resolution (≤1.8 Å) crystal structures in a pH range from 5.5 to 7, implying that an iron-bound persulfenate is energetically accessible in the CDO active site.92

Tyr157 L-Cys L-Cys His86 Cys93 Fe His140 Fe His88

His88 His86 His140

Figure 2. The overall reaction catalyzed by CDO (top); the Fe center of CDO with a bidentately bound Cys residue and a cysteinyltyrosine cross-link (PDB: 2IC1) (bottom left); and the cysteine persulfenate species captured in CDO crystals (PDB: 3ELN) (bottom right).

The catalytic function of the conserved Cys-Tyr cross-link near the iron active site in all mammalian CDOs has been subjected to intense studies. The presence of a Cys-Tyr cross-link only increases the catalytic efficiency by ~ 10 fold when compared with uncrosslinked mammalian CDOs.93 In bacterial CDOs, this cross-link is not present; the corresponding Cys residue in mammalian CDOs is generally replaced with a Gly residue. Interestingly, without the Cys-Tyr cross-link, bacterial CDOs have turnover rates similar to those of mammalian CDOs.94 The initial hypothesis for the function of the Cys-Tyr cross-link came from Rao and coworkers.83 Based on their L-Cys bound Fe(II)-CDO structure, they suggested that the cross-link could be used to stablize the iron center in a ferrous state and also prevent the leakage of the reactive oxygen species during catalysis. Based on steady-state kinetics, O2 consumption and product formation measurements, and EPR studies, Pierce and coworkers suggested that the presence of the Cys-Tyr cross-link increases both kcat and the O2/CSA coupling efficiency and also could bring Y157 into the right position to stabilize the substrate Cys binding geometry in the active site.95 Based on steady-state kinetics and the C93G CDO variant crystal structure with no cross-link, Jameson and coworkers observed that the cross-linked CDO decreases the optimal pH for the enzyme activity and extends activity to higher pH when compared with the uncross-linked CDO.96 They further proposed that the advantage of cross-link formation is not to directly change the active site structure and increase activity but to remove side reactions that could be caused by the free Cys93 thiolate at higher pH. Based on 21 high-resolution (< 1.6 Å) mammalian CDO structures, Karplus and coworkers concluded that the cross-link enhances CDO activity by positioning the Tyr157 hydroxyl to enable proper L-Cys binding, proper oxygen binding, and optimal chemistry.97

In addition to the function of the Cys-Tyr cross-link, the details on its biogenesis are largely unknown. Based on in vitro studies using purified recombinant protein along with rat liver lysate and in vivo studies using transfected hepatoma cells and intact rats, Stipanuk and coworkers concluded that the cross-link formation is dependent on binding of the cysteine substrate and the occurrence of catalytic turnover. This process is inefficient, requiring multiple turnovers to complete.93 By utilizing unnatural tyrosine incorporation, Liu and coworkers obtained fully uncross-linked human CDO structures with the incorporation of 3,5-difluoro-L-tyrosine (F2- Tyr157) in the presence and the absence of L-Cys.98 The authors even observed the Cys-Tyr cross- link formation in crystallo upon exposing the crystal of an anaerobically prepared fully uncross- linked Fe(II)-(F2-Tyr157)CDO-L-Cys complex to O2 (Figure 3), implying a potential C-F bond activation step. Very recently, the same group obtained crystal structures of fully uncross-linked 99 and mature human CDO bound with L-Cys and NO in the presence of F2-Tyr157. Based on these structures, the authors suggested that Cys93 is most likely the first target for oxidation during the iron- and O2-assisted Cys-Tyr cross-link formation.

Figure 3. Superposition of the substrate-bound mature F2-Tyr CDO (pink, PDB: 6BPV) and 100% uncross- linked structure (green, PDB: 6BPT). The figure is adapted from ref 98.

12 Scheme 5. Mechanistic hypotheses of the CDO-catalyzed reaction. Top: A mechanism featuring Fe(III)- superoxo and Fe(IV)-oxo species derived from spectroscopic and computational studies; Bottom: A mechanism featuring cysteine persulfenate species derived from crystallographic studies.

Based on many structural, spectroscopic and computational studies, two principal CDO reaction 81,83,91 mechanisms are proposed (Scheme 5). In the first mechanism, CDO binds O2 to form an Fe(III)-superoxo species that attacks the bound sulfur of L-Cys by the distal oxygen of the superoxide. This step forms an intermediate consisting of Fe, O2, and the cysteine sulfur (species C in Scheme 5), which undergoes heterolytic O-O bond cleavage to form an Fe(IV)=O intermediate. The ferryl species then supplies the second oxygen atom to sulfur to complete the reaction. The involvement of the Fe(III)-superoxo species has been demonstrated by Pierce and coworkers.100 By treating a substrate-bound Fe(III)-CDO with superoxide anions, a transient species with an optical absorption feature at 565 nm and integer spin EPR signal at g ~ 11 is formed rapidly. Spectroscopic simulations suggest that this intermediate is the Fe(III)-superoxo species with an S = 3 ground state. More importantly, the decay of this species leads to the formation of CSA, indicating this species is a kinetically competent intermediate for product formation. However, the involvement of the Fe(IV)=O has not been demonstrated experimentally. Recently, Jameson, de Visser, and coworkers provided the first experimental evidence of a short-lived intermediate observed by SF-Abs and Mössbauer spectroscopy.87 Along with computational studies, they suggested that the short-lived intermediate is most likely the species C in Scheme 5, although the Fe(III)-superoxo species cannot be entirely ruled out.

In the second mechanism, the cysteine persulfenate species observed in several CDO crystal structures is proposed to be the key intermediate in the CDO catalysis. In this mechanism,92 high- valent iron intermediates are not utilized. Rather, the initial cysteine oxidation occurs via sulfur attacking the Fe-proximal oxygen to form the crystallographically observed persulfenate species. The subsequent bond formation between sulfur and the Fe-distal oxygen generates a three- membered ring involving an S-O-O unit. O-O bond scission then completes the reaction. 3.4 Sulfoxide Synthases, EgtB and OvoA

ESH and OSH are two naturally occurring organothiols. Although their exact physiological role is not clear, it is believed that they are involved in relieving oxidative stress in cells due to their high redox potential.43,65,66 Very recently, Seebeck and coworkers have discovered the biosynthetic pathways for ESH and OSH.58,59 One of the interesting features in these pathways is that EgtB and OvoA catalyze installation of the thiol group onto to an imidazole carbon of TMH and His, respectively. Interestingly, in EgtB the oxidative sulfur carbon coupling occurs at C2 (ε position) of the imidazole moiety of TMH, while in OvoA it is at C5 (δ position) of the imidazole moiety of His. In addition, a sulfoxide moiety is also generated in both EgtB and OvoA catalysis to complete an overall 4-electron oxidation reaction. Thus, EgtB and OvoA reactions represent a new enzymatic strategy to install C-S bonds (Scheme 6).

Scheme 6. Native Reactions (left) and Alternative Reactions (right) Catalyzed by EgtB and OvoA.

EgtB and OvoA are NHMI O2 activating enzymes. Based on amino acid sequence, Seebeck and coworkers initially predicted the iron-binding motif to be a 2-His-1-Glu triad, the same with other non-heme iron enzymes, such as Fe/αKG enzymes.59 However, the first crystallographic study by Seebeck and coworkers on EgtB from Mycobacterium thermoresistibile (MtEgtB) revealed a 3- His motif as the actual iron-binding motif.101 Moreover, very different from other non-heme iron enzymes, EgtB adopts a two-domain structure consisting of a N-terminal domain homologous to zinc-dependent thiol-S transferases (DinB_2 domain) and a C-terminal domain similar to the copper-dependent formylglycine forming enzymes (FGE-like domain). The iron active site is located at the interface between the two domains. Based on bioinformatic analysis, Liu and coworkers also identified that some EgtB homologs contain an additional methyltransferase domain.102 In the first crystallographic study,101 the structures of MthEgtB in complex with TMH and iron and enzyme with manganese, γ-GC, N-α-dimethylated histidine (DMH) were solved (Figure 4). Both the Fe and Mn centers adopt a 6-coordinate geometry. The direct binding of the imidazole moiety of TMH and DMH through the Nε position and of γ-GC via the thiolate S to the metal center is revealed. In addition, the crystal structures also reveal that a tyrosine close to the C terminus (Tyr377) is positioned close to the metal center and establishes a hydrogen bonding interaction to a metal-bound water. This water coordination site is the potential O2 binding site. Thus, the position of Tyr377 implicates a role in EgtB catalysis (see below). Interestingly, Tyr377 is not strictly conserved in all known EgtB sequences. Very recently, Seebeck and coworkers solved crystal structures of an EgtB homologue from Chloracidobacterium thermophilum (CtEgtB).103 CthEgtB does not contain the corresponding Tyr377 found in MtEgtB in its sequence, but the crystal structures reveal that two different tyrosine residues close to the N terminus of the protein (Tyr93 and Tyr94) are positioned close to the presumed O2 binding site of the iron center. Furthermore, a large section of the rigid active site observed in MtEgtB is replaced in CtEgtB by two mobile active site loops that fold in a substrate-dependent fashion. Thus, there exists significant active site diversity among different EgtB homologs. So far, no crystal structure of OvoA has been reported in the literature.

Figure 4. Structure of the metal center in EgtB. PDB: 4X8D (DMH: dimethylated histidine).

Since the initial characterization of EgtB and OvoA, the search for a detailed mechanistic understanding of this unique C-S bond coupling reaction has been a key research topic. The substrate scope of EgtB is relatively narrow. All the characterized EgtBs exclusively use TMH as the sulfur acceptor in the catalysis. The sulfur donor can be γ-GC (for EgtB from Mycobacterium smegmatis, MsEgtB and MtEgtB)58,101 or cysteine (for Egt1 from Neurospora crassa and CtEgtB).102,103 OvoA can only use cysteine as the sulfur donor, but it has a much broader substrate scope with respect to the sulfur acceptor.104,105 Seebeck and coworkers discovered that the amino acid moiety of histidine is not essential for the OvoA catalysis.104 They also demonstrated that D- histidine can also support the OvoA reaction, but produces a mixture of 5-D-histidyl-L-cysteine sulfoxide and 2-D-histidyl-L-cysteine sulfoxide, suggesting the switch of the C-S coupling site from the native C5 to C2 on the imidazole moiety.104 By using histidine analogues with different methylation states on the amine group, Liu and coworkers also observed this switch of the regioselectivity of the C-S coupling site.105 These observations could result from the different binding modes of the histidine analogues. However, since no OvoA crystal structure is available, the exact reason is still unknown. Seebeck and coworkers further demonstrated that OvoA converts 2-fluoro-L-histidine to 2-fluoro-5-L-histidyl-L-cysteine sulfoxide with almost the same efficiency as the native reaction on L-histidine.104 This observation strongly suggests that a reaction intermediate containing an imidazole radical species is less likely, or at least is not rate determining. An important development in the understanding of the EgtB and OvoA mechanisms is the observation of CDO activity in both EgtB and OvoA. Liu and coworkers106 first demonstrated that OvoA not only catalyzes oxidative C-S coupling, but also generates the cysteine-oxygenated product, CSA (Scheme 6). The relative amounts of CSA formation can be tuned by histidine analogues with different methylation states. Seebeck and coworkers107 further demonstrated that a single point mutation (Y377F) converts MtEgtB to CDO by producing exclusively γ-GC oxygenated product (Scheme 6). This observation highlights the important role of Y377 in the EgtB catalysis. Interestingly, the CDO activity in EgtB and OvoA exclusively depends on the presence of histidine and its analogues, but they are not used as the substrate. Cysteine and γ-GC are bound to iron through the thiolate S in a monodentate fashion instead of bidentate mode as seen in CDOs. Recently, Liu and coworkers also demonstrated that the CDO and the oxidative C-S coupling reactivities in OvoA can be modulated by the tyrosine analogue.108

15 Based on biochemical data and computational modeling,109 the possible reaction mechanisms accounting for the native reactivity of OvoA are shown in Scheme 7. Similar mechanistic hypotheses with different regio-selectivity of C-S bond formation (C2-S formation of OvoA vs. C5-S formation of EgtB) can be used to explain the native reactivity of EgtB. All these mechanisms agree that the initial reactive intermediate should be the Fe(III)-superoxo species and the main difference after the decay of Fe(III)-superoxo species is the order of sulfoxide formation and C-S bond coupling. All the available experimental data seem suggest that the C-S coupling should precede the sulfoxide formation and the tyrosine residue close to the iron center plays an essential role in branching the catalytic reactivity between C-S coupling and cysteine dioxygenation.

Scheme 7. Mechanistic Hypotheses for OvoA-Catalyzed Reactions. Top: a mechanism involving an Fe(IV)=O intermediate with the formation of sulfoxide preceding the C-S bond coupling. Bottom: a mechanism with the formation of the C-S bond prior to the sulfoxide formation.

4. Ring-Cleaving Dioxygenases 4.1 Introduction

Ring-cleaving dioxygenases are involved in aerobic degradation of aromatic compounds, which is a critical step to maintain the global carbon cycle.4,110,111 Based on the mode of ring cleavage, there are two major types of enzymes: intradiol-cleaving dioxygenases that carry out ortho cleavage by

16 splitting the C=C bond on the aromatic substrate between the two hydroxyl substituents to form di-carboxylate products, and extradiol-cleaving dioxygenases that perform meta cleavage by cleaving the C=C bond adjacent to only one hydroxyl substituent to form a ring-cleaved product with one carboxylate and one aldehyde functional group at the opposite ends of the ring-cleaved product.4 In fact, these two types of enzymes are phylogenetically unrelated and possess different protein folds and distinct metal active sites. More importantly, they utilize different mechanisms of catalysis, namely metal-assisted O2 activation by iron(II) or manganese(II) centers of extradiol- cleaving dioxygenases vs. substrate-assisted O2 activation by iron(III)-containing intradiol- cleaving dioxygenases.1,2,4,9,11,14 Extradiol-cleaving dioxygenases are more versatile than their intradiol counterparts; they have broader substrate scope, use multiple protein scaffolds, and are involved in a variety of biological pathways.4 Some extradiol-cleaving dioxygenases are found in natural product biosynthetic pathways,112 but others may be associated with several severe diseases in humans, including the neurodegenerative disorder Huntington’s chorea and degenerative arthritis.113,114 In contrast, intradiol-cleaving dioxygenases have a narrow substrate scope with substrates limited to catechol, protocatechuate (3,4-dihydroxybenzoate) and 2-hydroxyquinol (1,2,4-trihydroxybenzene).4

The best characterized extradiol-cleaving dioxygenase is homoprotocatechuate 2,3-dioxygenase (HPCD).14 Two forms of the enzyme have been identified that use Fe(II) or Mn(II) as the metal center, respectively, to catalyze the cleavage of the C2-C3 bond of homoprotocatechuate (HPCA, 115 23) with the incorporation of both oxygen atoms from O2 into the product (Scheme 8 top). HPCD belongs to the type I family of extradiol dioxygenases (vicinal oxygen chelate superfamily).4,116 The best studied intradiol-cleaving dioxygenase is protocatechuate 3,4- dioxygenase (3,4-PCD) from Pseudomonas putida, which catalyzes the ring cleavage of protocatechuate (PCA, 3,4-dihydroxybenzoate, 25) with the incorporation of both oxygen atoms 4,117 of O2 into the two carboxylates formed in the ring-cleaved product (Scheme 8 bottom). Members of this family use ferric cofactors to activate O2 and perform catalysis, which is very different from all other NHMI enzymes where ferrous cofactors are generally utilized. Herein, some well-studied examples including HPCD and 3,4-PCD will be discussed to illustrate the structural and mechanistic differences of these two types of ring-cleaving dioxygenases and related enzymes.

Scheme 8. The distinct ring cleavage reactions catalyzed by the extradiol-cleaving dioxygenase HPCD (top) and the intradiol-cleaving dioxygenase 3,4-PCD (bottom). 4.2 Structural Considerations

17 Crystal structures of the iron-containing HPCD (Fe-HPCD) from Brevibacterium fuscum have been solved.118–121 The overall protein structure exhibits a homotetrameric architecture. Each monomer consists of four tandem βαβββ units, which are used to form an N-terminal domain and a C-terminal domain where the metal center is located. The iron-binding motif is the classical 2- His-1-carboxylate facial triad found in many NHMI enzymes. The substrate (HPCA) binds to the iron center in a bidentate manner via the two hydroxyl groups (Figure 5). Based on spectroscopic and crystallographic studies, one of the hydroxyl groups (C2 hydroxyl) is believed to be deprotonated while the other one (C3 hydroxyl) maintains its O-H proton.118,122 The iron coordination site trans to the Glu ligand is the potential O2 binding site. His200 and Tyr257 lie close to the iron center, interacting with the substrate. His200 also interacts with the O2 binding site. Mutagenesis studies have shown that these two residues play crucial roles during the enzyme catalysis (see section 4.3 for further discussion).

Crystal structures of several other extradiol-cleaving dioxygenases have also been solved.123–128 One of them is the manganese-containing enzyme from Arthrobacter globiformis (Mn-MndD).118 This enzyme catalyzes the same extradiol-type reaction by using HPCA as the substrate. Fe-HPCD and Mn-MndD have 83% sequence identity, and their crystal structures exhibit remarkable similarity (Figure 5), not only in the overall protein architecture but also in the metal center structure (both in the first and the second coordination spheres). More interestingly, the steady state kinetic parameters of the enzymes are also highly similar, implying that the enzyme efficiency is practically identical, despite the use of different metal ions with very different redox potentials.14 These findings have led to intense studies on the plausible reaction mechanisms of HPCDs and related enzymes (see section 4.3 for further discussions). A B His200

His155

H2O HPCA Fe

His214

Glu267

Tyr257

Tyr447 C D Tyr447

PCA His460 Fe

H2O

Tyr408 His462

Figure 5. Overlays of the overall protein structures (A) and the iron center structures (B) of the enzyme- substrate complex, HPCD-Fe(II)-HPCA (green, PDB: 4GHG), and MndD-Mn(II)-HPCA (grey, PDB: 1F1V). The iron center structures of 3,4-PCD in the absence (C, PDB: 2PCD) and in the presence (D, PDB: 3PCA) of the substrate, PCA.

Crystal structures of 3,4-PCD exhibit a dodecameric protein architecture composed of 12 copies of αβ heterodimers.129 The α- and β-subunits exhibit high amino-acid sequence and structural similarity, thus suggesting an evolutionary divergence from an ancestral homodimer. The core structures of both subunits are in the form of mixed β sheets, which are unique to the intradiol dioxygenases.4 The ferric center lies at the interface between the α- and β-subunits, and is coordinated with Tyr447, His462, Tyr408, His460, and a solvent hydroxide to form a distorted trigonal bipyramidal geometry (Figure 5).129,130 The binding of PCA displaces the axial Tyr447 and the equatorial hydroxide, leading to a five-coordinate ferric center with a vacant site trans to His460 (Figure 5).131 Both hydroxyl groups of PCA are ionized once bound to the ferric center.131,132 This is different from the bound HPCA in HPCD, where only one hydroxyl group of HPCA is deprotonated. This difference is likely due to the different Lewis acidities of trivalent and divalent metal centers that lead to the distinct reaction mechanisms of these two different enzymes (see section 4.4 for further discussions). 4.3 General Reaction Mechanism of Extradiol-Cleaving Dioxygenases.

Scheme 9 depicts the current working mechanistic model for the extradiol-cleaving 14 dioxygenases. It features an unique strategy to activate O2, where Fe(II) functions as a conduit to transfer an electron from the substrate to the bound O2 to form an SQ-Fe(II)-superoxo diradical species (where SQ is semiquinone). The radical recombination leads to the formation of an Fe(II)- alkylperoxo intermediate, the decay of which leads to C-O bond formation and O-O/C-C bond cleavage steps that eventually produce the desired ring-cleaved product.

Scheme 9. A Working Reaction Mechanism for HPCD.

The most important feature in this mechanism is the redox-inactive Fe(II) center. This is supported by the results of metal ion substitution experiments in HPCD and MndD.120 The metal-swapped Mn-HPCD (Mn(II)-substituted HPCD) and Fe-MndD (Fe(II)-substitued MndD) exhibit comparable catalytic activity and efficiency to those of the native Fe-HPCD and Mn-MndD. Furthermore, a Co(II)-substituted HPCD (Co-HPCD) also maintains catalytic activity with O comparable kcat to those of Fe-HPCD and Mn-HPCD, but exhibits a high Km for O2 (Km 2), which O suggests low O2 affinity, yet the contributions of other slow steps in the reaction to high Km 2 cannot be ruled out.133 The crystal structure of Co-HPCD shows that its active center is superimposable to those of Fe-HPCD and Mn-HPCD. In line with these observations, the overall O catalytic efficiency of O2 activation (kcat/KM 2) does not vary significantly among these metal- substituted HPCDs, suggesting that the activation energy for the key step is not directly related to the redox potential of the metal (the standard metal(III/II) potential of aqueous Co(II) is 1.15 V higher than that of Fe(II)).14 A reasonable explanation for this observation is that the metal center does not change oxidation state during this step, making its redox potential of secondary importance.

The best support for the current mechanistic model comes from crystallographic studies of Fe- HPCD. Lipscomb and coworkers have trapped three different intermediates in different active sites within the asymmetric unit of a single crystal of Fe-HPCD with a bound 4-nitrocatechol (4NC), a substrate analogue that is harder to oxidize than HPCA (Figure 6).119 This has been achieved by exposing the crystal to a very low concentration of O2 before freezing. One intermediate features O2 bound in a side-on manner to the iron with relatively long Fe-O bonds, suggesting the iron center remains ferrous (Figure 6A). In addition, the aromatic ring of 4NC is nonplanar, suggesting that an electron has been transferred from 4NC to the bound O2. Thus, this intermediate may represent the SQ-Fe(II)-superoxo diradical species (Scheme 9). The next intermediate features an alkylperoxo moiety bound to both the iron center and the C2 of the aromatic ring of 4NC (Figure 6B). The O-O bond is observed to be longer than that in the first intermediate (1.52 Å vs 1.31 Å), consistent with the alkylperoxo assignment. In the final intermediate, a ring-opened product complex is observed (Figure 6C). The same three intermediates as those of HPCD have also been found in crystallo in another ring-cleaving enzyme, homogentisate 1,2-dioxygenase (HGDO, see further discussion in section 4.5).134 Homogentisate is an isomer of HPCA, which has two hydroxyl groups para to each other on the aromatic ring instead of ortho. HGDO catalyzes a slightly different ring-cleaving reaction but may utlize a similar reaction mechanism as that of HPCD (Scheme 13). These results not only demonstrate that fully catalytic reactions can be performed in crystals, but also provide strong support for the current mechanistic model of extradiol-cleaving enzymes.14

20 His200

1.31 Å 1.52 Å

Tyr257 A B C

Figure 6. Intermediates (A and B) and the enzyme-product complex (C) observed in different subunits of the Fe-HPCD(4NC) complex found within the asymmetric unit of the crystal after reacting with O2 (PDB: 2IGA). Metal redox changes during HPCD reactions have been observed in spectroscopic studies on protein variants and substrate analogues. By using the H200N-Fe-HPCD variant with 4NC, two intermediates have been observed.135 Based on detailed Mössbauer and EPR characterizations, Münck, Lipscomb and coworkers concluded that the first intermediate (H200N-Fe-4NCInt1) is an Fe(III)-superoxide species. It exhibits a sharp g = 8.17 parallel mode EPR signal, suggesting an S = 2 ground electronic state (Figure 7). This ground state is formed by antiferromagnetic interactions between an S = 5/2 Fe(III) center with an S = 1/2 superoxide radical (supported by 17O hyperfine parameters detected by EPR and Mössbauer measurements, Figure 7). This is the first example of such an intermediate found in NHMI enzymes. Decay of this intermediate leads to formation of a second intermediate (H200N-Fe-4NCInt2). This new intermediate still has an S = 2 ground electronic state (Figure 7) and could be best described as a 4NC-SQ-Fe(III)-peroxo species. However, this species does not carry out ring cleavage. Peroxide is slowly released and the quinone product (not ring cleaved) is generated. The second electron from the SQ goes to the iron to restore Fe(II). A similar SQ-Fe(III)-peroxo species has been observed in the reaction of H200N-Fe-HPCD with the native substrate HPCA (H200N-Fe-HPCAInt1).136 This intermediate does attack the ring and further decays to another intermediate (H200N-Fe-HPCAInt2) with the iron center in its ferrous state. The second intermediate is likely to be the Fe(II)-alkylperoxo intermediate proposed in the current reaction mechanism. Metal redox changes in HPCD catalysis have further been observed in Mn-HPCD by using the native substrate HPCA and in Co-HPCD by using 4NC as the substrate (both for wild type and the H200N variant).137,138

137 In Mn-HPCD, the first intermediate generated within 0.015 s after O2 mixing with Mn-HPCD- HPCA complex could be best described as a Mn(III)-superoxide species based on its EPR parameters. Decay of this species gives rise to a second species with a very short lifetime, most likely a Mn(II) species. Further decay of this second species lead to the generation of product. In 138 Co-HPCD-4NC, upon exposure to O2, a high-spin (S = 3/2) Co(II) center is converted to an S = 1/2 species, which is best described as an S = 0 Co(III) coupled with an S = 1/2 superoxide. Thus, different from Fe-HPCD and Mn-HPCD, O2 addtion in Co-HPCD even causes a spin state change (high-spin to low spin conversion) together with an oxidation state change. For wild-type Co-

21 HPCD, this species further decays to the initial Co(II) center, while the H200N-Co-HPCD species is a dead-end complex. These metal redox changes observed in HPCD reconstituted with Fe(II), Mn(II), and Co(II) are inconsistent with the results from the metal ion substitution experiments and the current mechanistic model (Scheme 12), where the divalent metal center does not change redox state during the catalysis. It is possible that the actual electron transfer from the substrate to O2 happens in a stepwise manner, first from the metal to O2 and then from the substrate to the metal. In the native enzyme with the native substrate, the electron transfer steps may take place in rapid succession. It is also possible that the reaction of the metal(III)-superoxo species with the substrate occurs at a rate that is too fast to be detected.14

Figure 7. Left: parallel mode EPR spectra of H200N-Fe-4NCInt1 measured at 2 K (A), 9 K (B), and 10 K 17 Int2 with the use of O2, (C) and of H200N-Fe-4NC (D). Right: Mössbauer spectra of H200N-Fe(II)-4NC complex (A), of a sample containing H200N-Fe-4NCInt1 (red) and H200N-Fe-4NCInt2 (blue) (B), of H200N- Fe-4NCInt2 (C), and of a sample containing H200N-Fe(II)-4NC complex and H200N-Fe-4NCInt2. This figure is adapted from ref 135.

Computational studies have generally provided support for the overall mechanism shown in Scheme 9. However, differences can be seen in the details of the initial O2 activation steps. Using the iron center structure consisting of only the primary coordination sphere residues, Siegbahn and coworkers carried out initial DFT studies on HPCD.139,140 Their results suggested that the metal center acts as an electron conduit for the initial O2 activation. These studies also identified the specific roles for His200 and Tyr257 residues in the catalytic cycles. H200 facilitates the proton transfer from the monoanionic substrate to the bound superoxide, which occurs simultaneously with the electron transfer from the substrate to O2. Both H200 and Y257 residues direct the radical recombination of superoxide with the SQ ring and assist in the subsequent product formation. In more recent DFT work, Christian et al. used a larger structural model consisting of the iron center with both primary and secondary coordination sphere residues of Fe-HPCD.141 They found that changes in metal oxidation state are an important aspect of O2 activation by HPCD, and Fe(III)- superoxide species should be the more viable key intermediate for the HPCD-catalyzed reaction. They also pointed out that the size of the computational model influences the electronic structure

22 of the superoxide intermediate and the amount of electron donation from the substrate to the metal. Lai and coworkers applied QM/MM on the whole protein to further investigate the HPCD mechanism.142 Based on this large computational model, they found a species with an electronic structure possessing both Fe(III)-superoxide and SQ-Fe(II)-superoxo diradical characters. This species exhibits a lower activation barrier for the formation of the alkylperoxo intermediate than those from the Fe(III)-superoxide and the SQ-Fe(II)-superoxo species.

Very recently, Solomon, Lipscomb, and coworkers used NRVS and experimentally calibrated DFT calculations to define the structures of Int-1 and Int-2 in H200N-Fe-4NC HPCD and the crystallographically observed intermediates in the wild type Fe-HPCD with a bound 4NC.143 They concluded that H200N-Fe-4NCInt1 is the Fe(III)-end-on superoxo-catecholate species, consistent with the previous Mössbauer and EPR study by Münck, Lipscomb and coworkers.135 This species is competent in performing electrophilic attack on the catecholate. H200N-Fe-4NCInt2 is best described as an Fe(III)-end-on-hydroperoxo-SQ species (consistent with the computational study by Christian et al.), and the first wild-type crystallograhic intermediate is described as a side-on Fe(III)-hydroperoxo-SQ species (Figure 6A). However, both of these species are unreactive towards ring cleavage. The former is a dead-end species, while the latter can be converted to an isoenergetic reactive Fe(III)-end-on-superoxo-catecholate species via deprotonation by H200. Finally, the peroxo-bridged species seen in the crystallographic data (Figure 6B) is best assigned as an unprotonated delocalized Fe(III)-peroxo-SQ/Fe(II)-peroxo-quinone intermediate. It is important to note that the difference of electronic properties of 4NC and the native substrate HPCA may result in differences in the nature of key reactive species. Thus the end-on Fe(III)-superoxo- catecholate species is the reactive intermediate when 4NC is used, while a Fe(II)-superoxo-SQ species could be utilized as the key intermediate when HPCA is used. 4.4 General Reaction Mechanism of Intradiol-Cleaving Dioxygenases.

Scheme 10 depicts the current working hypothesis for the reaction mechanism of intradiol- cleaving dioxygenases, which has largely derived from the studies on 3,4-PCD and catechol 1,2- 9,131,132,144–147 dioxygenase. It features a unique strategy to activate O2, namely a concerted O2 addition to both C4 of the catechol and the high-spin (S = 5/2) Fe(III) center to form an Fe(III)- alkylperoxo intermediate. EPR and Mössbauer studies have shown that the iron center remains in the high-spin ferric state upon substrate binding.148,149 Thus the formation of the Fe(III)- alkylperoxo intermediate (Scheme 10, species D) requires a spin-forbidden two-electron transfer 9 from the singlet catecholate to the triplet O2. It is hypothesized that the spin-forbidden nature is circumvented by the trivalent iron-bound catechol, which may tautomerize to infuse Fe(II)- semiquinone character onto the catechol-bound Fe(III) center or acquire significant ketonized- character on the catechol to promote O2 addition (Scheme 10). Based on MCD studies of 3,4-PCD, Solomon and coworkers have concluded that the catecholate-bound Fe(III) center can be best described as a highly covalent Fe(III)-catecholate complex with an energetically high-lying doubly 150 occupied πop-sym (out-of-plane, symmetric) orbital from the catecholate moiety. This high-lying substrate orbital results in a low energy ligand-to-metal charge transfer (LMCT) band. This electronic feature provides a viable way to donate electrons from the substrate to an S = 1 O2 directly (through an α electron transfer) and indirectly (through a β electron transfer via the Fe(III) center), and the S = 5/2 Fe(III) center is thought to interact antiferromagnetically with O2 to yield 9 an Stot = 3/2 system during O2 addition. Spectroscopic and computational studies suggest that a conversion from a transient six-coordinate alkylperoxo intermediate to a five-coordinate species

23 is important to advance the reaction further.145,150 Next, O-O bond scission occurs and is followed by the re-establishment of a six-coordinate Fe(III) center and a simultaneous protonation of the proximal oxygen of the alkylperoxo moiety (Scheme 10, Species E). The source of the proton has not been identified, but it could be a solvent molecule or Tyr447, which are also the two candidates for the sixth ligand of Fe(III). O-O bond scission further leads to migration of one oxygen atom into the subtrate ring to form an anhydride (Scheme 10, Species F). Attack of the iron-bound hydroxide on the anhydride then cleaves the substrate ring to form the product complex. The release of the product completes the reaction cycle.

Scheme 10. The Current Working Reaction Mechanism for 3,4-PCD.

Although transient state kinetic studies have identified intermediates in the reaction catalyzed by 3,4-PCD, the unfavorable kinetics of their interconversion have not permitted any in-depth spectroscopic characterization of these intermediates.144,151–153 Recently Lipscomb and coworkers have characterized two reaction intermediates in the 3,4-PCD reaction in crytallo by using 4- fluorocatechol (4FC) as the substrate.154 4FC is a substrate analog that slows the 3,4-PCD reaction at several steps throughout the reaction cycle. The two intermediates revealed in this study are the Fe(III)-alkylperoxo intermediate and the Fe(III)-anhydride intermediate (Figure 8). The structures of both intermediates are almost identical to the Fe(III)-alkylperoxo species proposed from computational studies. The crystal structure of the Fe(III)-alkylperoxo intermediate features a peroxo moiety with an O-O bond distance is 1.5 Å, connecting the iron center and C4 of the iron- bound catecholate. Tyr447 is found in both metal-bound and metal-unbound orientations at equal occupancy. In the crystal structure of the Fe(III)-anhydride intermediate, the O-O bond is cleaved with one oxygen atom migrated into the ring of the substrate and the other oxygen atom remaining

24 metal-bound. These in crystallo observed intermediates thus provide strong experimental evidence to validate the reaction mechanism depicted in Scheme 10.

Figure 8. Iron center structures of the intermediate species observed upon reacting 3,4-PCD with 4FC and O2 at pH 6.5 and 8.5. (A) Bonding environment of the Fe(III)-alkylperoxo intermediate; (B) Bonding environment of the Fe(III)-anhydride intermediate. This figure is adapted from ref. 154. 4.5 Other Ring-Cleaving Dioxygenases.

In addition to the extradiol-cleaving and the intradiol-cleaving dioxygenases described above, there is a third group of ring-cleaving dioxygenases, with substrates that do not contain vicinal dihydroxyl groups on the aromatic rings (for a more detailed review, please refer to ref 13). Some examples and the corresponding reactions are listed in Scheme 11.13 Among them, HGDO127 and 3-hydroxyanthranilate 3,4-dioxygenase (HADO)155–157 use the classical 2-His-1-carboxylate iron binding motif to coordinate iron, while gentisate 1,2-dioxygenase (GDO),158,159 1-hydroxy 2- naphthoate dioxygenase (HNDO), salicylate 1,2-dioxygenase (SDO),160,161 and 5-chloro-gentisate 1,2-dioxygenase (CGDO) use a 3-His coordination motif to bind iron. The following discussion focuses on HGDO and GDO.

Scheme 11. Examples of Ring Cleaving Enzymes with 2-His-1-Carboxylate and 3-His Iron Binding Motifs. (A) homogentisate dioxygenase (HGDO) and (B) 3-hydroxyanthranilate 3,4-dioxygenase (HADO) as well as their 3-His coordinated counterparts (C) gentisate dioxygenase (GDO), (D) 1-hydroxy 2-naphthoate dioxygenase (HNDO), (E) salicylate 1,2-dioxygenase (SDO) and (F) 5-chloro-gentisate 1,2-dioxygenase. This figure is adapted from ref 13.

Due to the similar extradiol-cleaving-type reaction outcomes, it is generally believed that the reaction mechanisms of HGDO and GDO would follow closely to that of extradiol-cleaving dioxygenases, such as HPCD. Early biochemical studies have established that the ferrous iron is the catalytically competent metal cofactor for GDO.162–164 The 3-His iron-binding motif was revealed by crystal structures of GDO from E. coli and from Silicibacter pomeroyi.158,159 However, no substrate-bound crystal structure has been reported. The substrate-binding configuration of 17 17 GDO was first revealed through EPR studies on a Fe-NO complex using OH2- and O-labeled gentisate analogues.163 It was concluded that gentisate binds the ferrous center in a bidentate fashion through its carboxylate and 2-OH moieties. This binding configuration was further confirmed by the crystal structures of substrate-bound SDO, a related enzyme to GDO.160,161 The proposed reaction mechanism for GDO is shown in Scheme 12, which closely follows the reaction mechanism of HPCD illustrated in Scheme 9. One difference is that gentisate binds to the Fe(II) center in a di-ionized form, which is different from HPCD where HPCA binds to the Fe(II) center in a mono-ionized form. Based on QM/MM calculations on SDO, Roy et al. suggested that the di-

26 ionized salicylate exerts a strong covalent interaction with the Fe(II) center, similar to the binding of PCA to the Fe(III) center of 3,4-PCD. This strong interaction leads to a Fe(II)-O2 adduct that is best described as a Fe(II)-superoxo species with some radical character on the salicylate, much 165 like the SQ-Fe(II)-superoxo species proposed for HPCD. Moreover, O2 activation in SDO happens without the assistance of a proton source.

Scheme 12. Proposed Reaction Mechanism of Gentisate Dioxygenase (GDO).

HGDO, an enzyme highly similar to GDO, catalyzes the ring-cleaving reaction of homogentisate but uses a 2-His-1-carboxylate motif to bind the iron, instead of a 3-His motif observed in GDO. This was first revealed by the crystal structures of human HGDO in the apoprotein and the Fe- bound state.127 Based on the similarity of the reaction catalyzed by HPCD, GDO and SDO, Siegbahn and coworkers166 proposed a bidentate-binding configuration of homogentisate to the ferrous center and a possible reaction mechanism for HGDO. However, a recently reported substrate-bound crystal structure of HGDO by Dobbek and coworkers features a monodentate binding configuration of homogentisate to Fe(II)-HGDO through the 2-OH moiety of the aromatic ring (Figure 9).134 The substrate carboxylate, although initially proposed to bind to the iron center, actually interacts with Tyr346 and several solvent molecules through hydrogen bonds, thus preventing it from coordinating with the iron center. More interestingly, Dobbek and coworkers also observed several key intermediates in crystals, which can be best described as the SQ-Fe(II)- superoxo species, the Fe(II)-alkylperoxo intermediate and the ring-cleaved product bound species. These observations parallel to those reported by Lipscomb and coworkers for Fe-HPCD. Based on these experimental observations, Dobbek and coworkers proposed a reaction mechanism of HGDO that mirrors the HPCD mechanism (Scheme 13). Using QM/MM calculations, Lai and coworkers provided further insights into HGDO catalysis.167 Their computational results suggested that, if the 2-OH group binds to the Fe(II) as the conjugate base, then the attack of the SQ-Fe(II)-superoxo species on the substrate is rate-limiting; but if the 2-OH group binds to the Fe(II) without loss of the OH proton, then the decay of the Fe(II)-alkylperoxo intermediate becomes rate-limiting. Moreover, based on computationally optimized geometries, they proposed that the 2-OH group is more likely to remain protonated in the crystal structure.

27 A B

Tyr346 His288

homogentisate 1.35 Å H2O His331 Glu337

C His367 D

1.57 Å H2O

Figure 9. Iron active site structures of (A) HGDO-Fe(II)-homogentisate complex (PDB: 4AQ6), (B) and (C) the intermediate species observed upon reacting HGDO with homogentisate and O2, and (D) the ring cleaved product species (PDB: 3ZDS).

Scheme 13. Proposed reaction mechanism for homogentisate dioxygenase (HGDO).

28 5. Carotenoid Cleavage Oxygenases 5.1 Introduction

Carotenoid cleavage oxygenases (CCOs), along with closely related stilbene cleavage oxygenases (SCOs), are a family of NHMI enzymes that selectively cleave C=C double bonds in large conjugated polyeneic systems (carotenoids or stilbenes), producing aldehyde or ketone products. Natural products derived from carotenoid cleavage are ubiquitously distributed in all major domains of life,168 including animals that cannot synthesize carotenoids and must obtain them from diet or symbioitic microorganisms. In addition, proteins of this family such as RPE65 and NinaB have also been found to catalyze Z-E isomerization with or without double bond cleaving activity (Scheme 14). The important and diverse physiological functions of carotenoid derivatives,169 for example in vision, immunology, cancer, light harvesting, pathogen virulence, and pigmentation, have stimulated great interest in the characterization of CCOs and mechanistic studies as well.

Scheme 14. Examples of CCO-Catalyzed Reactions. β-CD: carotenoid oxidative cleavage. NOV1/2: stilbene oxidative cleavage. NinaB: carotenoid oxidative cleavage and Z-E isomerization. RPE65: non- oxidative ester cleavage and isomerization.

5.2 Structural Considerations

Known CCOs (including SCOs) have a seven-bladed β-propeller overall architecture, where the mononuclear iron center is coordinated by four histidine residues located around the central axis.169–176 Fe(II) is required for enzymatic activity, and a distorted octahedral geometry is assumed, with the two non-protein ligand positions cis to each other. In some CCO structures, one of these sites is blocked by a threonine or valine residue, making it inaccessible for large ligands.

29 In most known CCO crystal structures, dioxygen is not present. However, it was observed to bind to the Fe atom (Figure 10) in an end-on manner in a structure of VP14172 and in a side-on manner in a structure of NOV1.173 Interestingly, the VP14 structure was obtained without the presence of substrate, while in NOV1, O2 binding was detected in 3 situations: with a substrate, with one of the cleavage products, or without a substrate, product or inhibitor. In the structure of the NOV1 ternary complex with the substrate resveratrol and O2, the scissile C=C double bond was found to be directly facing the iron-bound dioxygen, roughly perpendicular to the O-O bond. Nitric oxide binding EPR experiments173,177 detected Fe-NO adducts with and without the presence of substrates. However, NO has a higher affinity for iron relative to dioxygen, and its binding is not conclusive evidence for the lack of a substrate-gated dioxygen binding step, which is common in many other families of iron oxygenases.

Not surprisingly, the substrate does not appear to directly participate in the first coordination sphere of iron. Different CCOs may have single-ended (e.g. NOV1) and double-ended (e.g. apocarotenoid oxygenase, ACO) substrate binding cavities. It has been proposed that the tunnel near the central axis, perpendicular to the propellers, tends to allow entry and exit of soluble substrates and products, while another possible tunnel, opening near hydrophobic patches on the surface, allows extraction of hydrophobic substrates from the membrane and their dissociation.173 The presence and absence of either tunnel may reflect the solubility of substrates and products. CCOs are typically highly regio-specific with regard to the double bond cleaved, and selective for certain chemical features of the substrate, but they also tend to show certain degrees of substrate promiscuity, allowing multiple substrates or analogues.169,173,177,178

Thr121 His412 O2

His218 His167 Fe His437 Fe

O2 OH

Resveratrol His476 His298 His590 His284

Figure 10. Dioxygen-bound active-site structures of NOV1 (left, PDB ID 5J54, with the substrate resveratrol) and VP14 (right, PDB ID 3NPE).

30 5.3 General Mechanism

The name CCO is often used interchangeably with carotenoid cleavage dioxygenase (CCD). Indeed, there has been an important debate on whether CCOs act as monooxygenases or dioxygenases. Isotope labeling experiments had yielded results consistent with both possibilities, with mechanisms proposed as shown in Scheme 15.

The monooxygenase hypothesis proposes an epoxide intermediate, which opens with a solvent water. The diol is further oxidized by an Fe(IV)=O species to cleave the C-C bond. The dioxygenase hypothesis utilizes Fe(III)-superoxo and Fe(II)-peroxo species as the oxidant, possibly forming a dioxetane. A quantum chemical study comparing two mechanisms slightly favored the dioxetane mechanism but could not rule out the epoxide mechanism.179 However, recent evidence has been mounting that favors the dioxygenase mechanism, as more exquisite experimental designs were applied to account for solvent oxygen exchange.180–182

The tertiary amine abamine was found to be an inhibitor of 9’-cis-epoxycarotenoid dioxygenase (NCED).183 While this experiment is not definitive, it lends credence for a substrate carbocation intermediate, which can be stabilized by the polyene system. It is proposed that the initial Fe(II)- O2 adduct, in equilibrium with an Fe(III)-superoxo complex, extracts an electron from the polyenic substrate to form a Fe(II)-peroxo-substrate cation. The substrate cation can then form a dioxetane by recombining with the peroxide anion, leading to eventual incorporation of both dioxygen atoms into the product. Alternatively, an acid-catalyzed Criegee rearrangement mechanism would allow possibilities of either one or two of the dioxygen atoms to be incorporated into the products, due to the addition of another water molecule.

Dioxygenase activity aside, the Z-E isomerization reaction is also of physiological importance and has attracted research attention. It has been found that the RPE65 isomerase activity does not require dioxygen,182 and a mechanism with Fe acting as a Lewis acid has been proposed.184 However, such a mechanism does not necessarily explain the reactivity of the isomerase- oxygenase NinaB, which might adopt an isomerase mechanism with reversible formation of a substrate radical that enables the rotation of the C-C bond.180

Scheme 15. General mechanism for CCOs. Oxygen atoms from dioxygen are indicated in bold. The current consensus favors the dioxygenase pathway. Note that the Criegee rearrangement route can incorporate solvent as well as dioxygen water into the product.

6. Lipoxygenases 6.1 Introduction

Lipoxygenases (LOXs) are a family of dioxygenases that catalyze the hydroperoxidation of polyunsaturated fatty acids. First discovered in soybean, they are currently known to be expressed in plants, animals, fungi, protists and prokaryotes, but not in archaea.185,186 The typical known function of LOXs is the anabolism (synthesis) of physiologically functional metabolites, as opposed to simple catabolism (degradation) of fatty acids for energy as intuition might suggest. LOX-generated fatty acid peroxides, along with cyclooxygenase (COX) products, are called eicosanoids (20-carbon lipids) or more generally oxylipins.187 These products are used in a number of biological pathways, such as plant chemical defense, environmental stress response, mammalian cellular proliferation and differentiation, as well as pathophysiological processes like inflammation and cancer progression. As such, LOXs have attracted considerable research interest as a pharmaceutical target and a biotechnological tool. LOX is also a prototypical system for studying enzymatic regio- and stereo-selectivity, C-H activation, and the proton-coupled electron transfer (PCET) process.

32 6.2 Structural Considerations

The substrates of LOXs are fatty acids containing one or more (1Z,4Z)-pentadiene units (Scheme 17). They abstract a hydrogen atom at the bis-allylic carbon (“C-3”), forming a radical before attaching a dioxygen at either C-1 or C-5. As is expected for producing signaling molecules, the regio- and stereo-selectivity is tightly controlled for both the hydrogen abstraction and the dioxygen insertion.185,187–189 The conventional nomenclature for LOXs reflects such selectivity, designating the position (and possibly chirality) of dioxygen insertion into arachidonic acid for mammalian LOXs, and into linoleic acid for plant LOXs. For example, a plant 13S-LOX inserts the dioxygen at the C-13 position of linoleic acid with S stereochemistry.

The majority of currently-known LOXs requires a non-heme mononuclear iron for activity, but more recent studies have also reported fungal LOXs utilizing manganese. The latter enzymes exhibit subtle differences in structure and reactivity while still sharing sequence and structure similarities with Fe-LOXs,.190

Most LOXs have a two-domain structure. The N-terminal β-barrel domain is involved in membrane binding and regulation of activity. The active site, along with the substrate binding pocket, is located in the α-helix-rich C-terminal domain. Structural research has revealed that in Fe-LOXs, Fe adopts a distorted octahedral geometry with five ligands from amino acid residues, in addition to a sixth hydroxide or water ligand, as shown in Scheme 16. In soybean LOX-1191 there are 3 histidine ligands, 1 asparagine ligand, and a carboxylate ligand from the C-terminal isoleucine. The Asn694 Oδ ligand is 2.85 Å from the iron. The human 5-LOX has a very similar first coordination sphere.192 The LOX 2 from a Cyanothece cyanobacteria193 also has the same ligand residues, but the Asn453 Oδ is 2.48 Å from the iron. In rabbit 15-LOX194, the ligands are instead 4 histidines plus the carboxylate of C-terminal isoleucine. His545 occupies the position corresponding to Asn694 of soybean LOX-1, but with Fe-N distance of 2.29 Å. His545 coordinates to Fe via Nδ, distinct from other histidine ligands that coordinate to Fe via Nε. In either case, the carboxamide Nδ of the “weak” asparagine or the Nε of the corresponding histidine can serve as a hydrogen bond donor, with potential acceptors in the close vicinity.

Scheme 16. First coordination spheres of Fe in soybean LOX-1 (left, PDB 3PZW) and rabbit 15-LOX (right, PDB 2P0M), highlighting the “weak” ligand.

While iron is at the Fe(II) redox state in the as-isolated enzyme, the active form of LOXs has been found to exhibit a charge transfer band at 350 nm195 and a high-spin (S = 5/2) Fe(III) EPR signal.ref A K-edge XAS study shows a six-coordinate Fe(III) center with one short (1.88 Å) Fe-O bond, indicating a hydroxide (OH-) ligand and an average distance of 2.11 Å for the other ligands.196 The discrepancies with the long Fe-ligand distance determined by crystallography probably indicate

33 mechanisms switching the Fe geometry between five- and six-coordination via second coordination sphere interactions.197 6.3 General Mechanism

Unlike most other NHMI enzymes discussed in this chapter, LOXs catalyze the reaction via substrate activation rather than dioxygen activation, similar to intradiol-cleaving dioxygenases (see Section 4.4).5 The mechanism consists of four elementary reactions: hydrogen atom abstraction, radical rearrangement, dioxygen insertion, and radical reduction, as illustrated in Scheme 17.

Upon binding of the fatty acid substrate, the reaction is initiated by the Fe(III)-OH unit abstracting one of the two doubly allylic hydrogen atoms of the substrate stereospecifically via a PCET process, with the proton transfer (PT) to the hydroxide ligand, and the electron transfer (ET) to the Fe(III). The Fe(III)/Fe(II) reduction potential in LOX is unusually high at 0.57-0.68 V, estimated by testing electron transfer rates from a series of substituted catechols to the LOX Fe(III) 198,199 200 center. Experiments with deuterated substrate revealed an unusually large KIE on kcat (kH/kD = 81) and a weak temperature dependence with an activation energy is only 2.1 kcal/mol. Substitution of hydrophobic amino acid residues alter the KIE and its temperature dependence. Significant computational and experimental efforts22,201 have been invested to achieve an understanding of this observation, highlighting the importance of non-adiabatic vibronic coupling to enable effective hydrogen tunneling in the PCET process.

After the formation of the delocalized pentadienyl radical, dioxygen is inserted into the antarafacial side of the leaving hydrogen. Structural studies have revealed many features that give rise to the regio- and enantio-selectivities of this step.185 Hydrophobic or charged residues at the bottom of the fatty acid binding pocket can affect whether it aligns in a “head-first” or a “tail-first” manner. Some LOXs have putative cavities that allow dioxygen to access specific positions of the bound substrate, and/or bulky residues that block the O2 access.

Nature may employ other possible strategies for selectivity as well. Amino acid residues in the binding pocket could alter the electron distribution of the pentadienyl radical to favor a specific product. The binding pocket could distort the planarity of the radical, forcing it to focus the electron density at a certain carbon. If the pentadienyl radical oxygenation is reversible, specificity can be achieved by stereo-selective peroxy radical reduction. Different LOXs may employ different mechanisms, and a combination of these factors is possibly required to achieve the high selectivity exhibited by LOXs.

Interestingly, LOXs may also exist in fusion proteins that incorporate other iron-dependent active centers catalyzing a cascade of reactions. For example, the first such protein discovered is a coral peroxidase-lipoxygenase, which contains a C-terminal 8R-LOX domain and an N-terminal heme peroxidase domain with a catalase fold.202,203 It produces an unstable allene oxide that may be converted into a cyclopentenone product in subsequent reactions.

Scheme 17. General Reaction Mechanism for Lipoxygenases.

7. Rieske Oxygenases 7.1 Introduction

Rieske non-heme iron-dependent oxygenases were previously called arene dioxygenases because the earliest discovered members of this family were bacterial enzymes for the early steps of biodegradation of aromatics,5,204–206 with both atoms of the dioxygen incorporated into the products. However, with more members of this enzyme family discovered, the old name no longer reflects their chemical versatility.

In addition to arene cis-dihydroxylation207, the current knowledge of enzymatic activities of Rieske oxygenases has expanded to include: N- and O-dealkylation,208–214 N-oxidation,215,216 mono- hydroxylation of aliphatic217–222 and aromatic223–225 C-H bonds, oxidative carbocyclization226–229 and heterocyclization230–232, decarboxylative hydroxylation,233 C=C double bond formation,234–238 and C-S bond cleavage.239 The above is by no means an exhaustive list, and some examples are shown in Scheme 18. They are now known to exist in many kingdoms of life, in both anabolic and catabolic pathways with aromatic and non-aromatic substrates, and both monooxygenase or dioxygenase-type reactions may be catalyzed by them.19,240,241 Therefore, Rieske oxygenase is a

35 more appropriate name for this group of enzymes, since it refers to a common structural feature, namely the Rieske iron-sulfur cluster, that is shared by all members of this category.

Scheme 18. Examples of Rieske Oxygenase-Catalyzed Reactions. Bold O atoms show oxygen incorporation in products. Bold C-C bonds (except for wedge bonds indicating stereochemistry) show new

36 bond formation. Exemplified types of Rieske oxygenase-catalyzed reactions include: NDO, arene cis- dihydroxylation; PrnD, N-oxidation; Stc2, N-demethylation; KshAB, C-H hydroxylation; RedG, oxidative carbocyclization; MarC, hydroxylation decarboxylation; AmbP, desaturation; MSAMO, C-S bond cleavage.

The characteristic Rieske-type [2Fe-2S] cluster is anchored to the protein via 2 His and 2 Cys residues. This type of cluster ligation, first discovered in the cytochrome bc1 complex (not a Rieske oxygenase),242 is distinct from the 4 Cys and the mitoNEET (3 Cys, 1 His) types of [2Fe-2S] clusters. Rieske oxygenases typically function as two- or three-component systems. The “terminal” oxygenase harbors the Rieske cluster, as well as the catalytic non-heme iron center. The system also requires a flavin-dependent ferredoxin reductase, and an additional ferredoxin in the case of three-component systems.204,206,243 An electron source such as NAD(P)H is also required for enzymatic activity. 7.2 Structural Considerations

Most of current structural and mechanistic knowledge of Rieske oxygenases stems from studies on various arene dioxygenases, and other types of reactions largely remain to be explored. Naphthalene dioxygenase (NDO), the archetypical Rieske dioxygenase, has an oxygenase protein that consists of the catalytic α- and the structural β-subunits. Crystallography reveals an α3β3 hexamer. The Rieske cluster from one α subunit is situated far away (~ 45 Å) from the non-heme iron center of the same subunit, but in the closer proximity (~ 12 Å) to the non-heme iron center from a second α subunit.243 This pair of metal centers from two monomers are within reasonable distance for electron transfer (ET), and are considered to form one functional unit. The subunits loop around head-to-tail to form a 3-fold rotationally symmetric overall structure. There are also Rieske oxygenases without the β subunit, assuming an αn (n is typically 3, 4 or 6) overall quaternary structure instead.204,243

A structure for a typical active site is shown in Figure 11.244 The non-heme iron, which is Fe(II) in the resting state of the active enzyme, has a distorted octahedral coordination geometry. The protein provides a 2-His-1-Asp “facial triad”, where the Asp carboxylate is a bidentate ligand, as opposed to monodentate in typical facial triad structures. The lack of second coordination sphere hydrogen bonding with this carboxylate is likely the reason for this binding mode.245 The other two positions can be occupied by water, substrate, product or dioxygen. Dioxygen is found to bind to the metal in a side-on manner.244,246

37 Cys101 Cys81

S Asp362’ S Fe His83 His104 His213’ Fe

O2 His208’

Asp205’

Indole

Figure 11. Active site of a Rieske oxygenase that includes both the Fe2S2 cluster and the monoiron catalytic center. Also shown are the bound substrate and dioxygen. The residue numbering (apostrophes denote another subunit) is from Pseudomonas putida NDO, PDB ID 1O7N.

The cluster iron distal to the mononuclear iron center is ligated by the two cysteines, and the 2+ proximal cluster iron by the two histidines. The cluster cycles between the [Fe2S2] (oxidized) + and [Fe2S2] (reduced) states, with a typical reduction potential of -0.15 to -0.05 V, which is higher than the typical 4-Cys cluster (-0.2 to -0.4 V) but lower than the Rieske clusters in the cytochromes (+0.15 to +0.49 V).247,248

A conserved aspartate residue (Asp205’ in Figure 11) serves as a bridge between one of the cluster histidine ligands and one of the mononuclear iron histidine ligands, with its carboxylate acting as a hydrogen bond acceptor. It is proposed that the Asp and the hydrogen bond network act as a conduit for ET. 2H Mims ENDOR experiments with the substrate-bound NDO-Fe(II)-NO adduct have been performed to estimate the distance between the electron spin center (Fe(II)-NO, S = 3/2) and substrate deuterons by measuring their dipolar hyperfine interaction.249 The data suggest that the reduction of the Rieske cluster shifts the balance between two substrate binding conformations towards the “B” conformation, in which the closest deuteron moves by ~ 0.5 Å away from the mononuclear iron, possibly allowing O2 access. Structural studies have also revealed exquisite conformational changes around the mononuclear iron, where the aspartate and histidine ligands move to adopt a geometry more accommodating for dioxygen.250 7.3 General Mechanism

As hinted in the structural section, NDO adopts an ordered mechanism for substrate binding, cluster reduction, and dioxygen binding. Single turnover experiments show that a reduced Rieske cluster, as well as substrate binding, is required for the oxygenase activity, and one mononuclear iron and one Rieske cluster are oxidized per product molecule formed -- a two-electron stoichiometry.251 The electron transfer proteins (reductase and ferredoxin) thus deliver, twice per catalytic cycle, an electron from an electron source to the non-heme iron via the Rieske cluster. The ferrous iron is in equilibrium between six- and five-coordinate geometry. Substrate binding,

38 along with a reduced Rieske cluster, biases it towards five-coordinate geometry conducive to dioxygen binding.252

Mechanistic studies have explored possible pathways for the dioxygenase reaction in terms of reactive intermediate species generated after dioxygen activation.19,206 In one model, the delivery of an electron from the cluster, protonation, and loss of a water ligand lead to a ferric hydroperoxide complex. A “peroxide shunt” pathway corroborates this Fe(III) hydroperoxide 253,254 mechanism. The fully oxidized enzyme can generate the product with H2O2 (instead of O2 and two electrons), incorporating both peroxide oxygen atoms into the product, albeit at a much slower rate. Nuclear resonance vibrational spectroscopy (NRVS) gives rise to a vibrational spectrum consistent with the DFT-simulated spectrum of side-on Fe(III)-η2-hydroperoxide complex, as opposed to those of side-on peroxide and end-on hydroperoxide complexes.255

However, a recent study of the benzoate 1,2-dioxygenase (BZDO) using the native substrate and its fluoro-substituted analogues has found that the rate constant for electron transfer from the Rieske cluster is strongly correlated with the electron density on the substrate carbon closest to the iron.256 Therefore, the substrate participates in the reaction before an electron is transferred from the Rieske cluster. The initial Fe-O2 adduct is proposed to be a Fe(III)-superoxide, which subsequently attacks the substrate and extracts an electron to form a Fe(III)-peroxide-aryl radical intermediate. The electron transferred from the Rieske cluster stabilizes this intermediate and facilitates O-O bond cleavage. An investigation with salicylate 5-hydroxylase, a Rieske monooxygenase catalyzing aromatic hydroxylation, has found a similar dependence of electron transfer rate on substrate substitutions, suggesting a similar Fe(III)-superoxo intermediate attacking the electron-rich arene ring.257

Investigations using radical clocks258 and 18O isotopic labeling259 implicate substrate radicals in the mechanism. However, different possibilities exist for the formation of such radicals: The Fe(III)-superoxo attack on the substrate can produce a substrate radical before electron transfer from the Rieske cluster as mentioned above. The Fe(III)-hydroperoxide intermediate can form radicals as well. It can attack the substrate first to form the radicals via O-O homolysis, yielding a Fe(IV)=O(OH) species (formally equivalent to Compound II of P450 catalysis) and a substrate radical; alternatively, it can first reorganize into a Fe(V)=O(OH) species (formally equivalent to Compound I in P450) via O-O heterolysis, before extracting an electron from the substrate. It is noteworthy that in the Fe(III)-hydroperoxide pathway, the possible highly reactive oxidants Fe(V)=O(OH) or Fe(IV)=O(OH) operate at +1 higher Fe valence than usually found in other non- heme mononuclear iron oxygenases (Fe(IV)=O and Fe(III)-OH). Such high-valent iron species might be the oxidant needed for Rieske oxygenases to activate strong C-H bonds, such as in aliphatic hydroxylation and carbocyclization.206

Scheme 19. Possible mechanistic routes for cis-dihydroxylation of arenes (represented by a benzene) catalyzed by Rieske oxygenases. Right: Superoxide pathway. Down: Hydroperoxide pathway, including possibilities of heterolytic and hemolytic breakage of the O-O bond.

In summary, possible routes of the catalytic cycle are presented in Scheme 19 with regard to the considerations above: the superoxide256 and the hydroperoxide19,205,206 pathways. In NDO, these possible “branching paths” converge at a Fe(III)-cis-diol product complex, from which the product is not released until another electron is transferred to the mononuclear iron from the Rieske cluster.260

8. Concluding Remarks

The catalytic versatility of co-substrate-independent NHMI enzymes rivals that of co-substrate- dependent NHMI enzymes; together they constitute quite a large enzyme family in nature. Such a broad reactivity exhibited by NHMI enzymes has only been observed for another family of O2 activating enzymes, namely the cytochrome P450 enzyme family.261 New members of NHMI enzymes are being discovered at a surprisingly fast pace in recent years. This progress not only provides a superb opportunity for (bio)chemists to gain detailed understanding on nature’s repertoire in utilizing O2 for chemical transformations, it also provides inspirations to develop these enzymes into valuable tools for chemical synthesis and biological pathway control. Mechanistic studies and bioengineering studies of NHMI enzymes will complement each other in the near future to truly harvest the power of evolution in enzyme catalysis.

40 9. Acknowledgements

The preparation of this chapter was supported by grants from the National Institute of Health (NIH GM125924 to Y.G. and GM127588 to W.-c. C.) and the National Science Foundation (NSF CHE- 1654060 to Y.G.).

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