Sulfur-Ligated, Oxidative Nonheme and Related Complexes Jesse B Gordon and David P Goldberg, Department of Chemistry, The Johns Hopkins University, Baltimore, MD, United States

© 2020 Elsevier Inc. All rights reserved.

1 Introduction 1 2 Isopenicillin N Synthase 2 2.1 Introduction 2 2.2 Structure and Function 2 2.3 Mechanism 3 3 Thiol Dioxygenases 7 3.1 Introduction 7 3.2 Structure and Function 7 3.3 Cysteine Dioxygenase Mechanism 10 3.4 ADO/3MDO/MSDO—The Other Thiol Dioxygenases 13 4 Sulfoxide Synthases 15 4.1 Introduction 15 4.2 Structure and Function 16 4.3 Mechanism 17 5 Persulfide Dioxygenase 19 5.1 Introduction 19 5.2 Structure and Function 20 5.3 Mechanism 21 6 Synthetic Model Complexes 22 6.1 Sulfur Oxygenation 23 6.2 Fe/O2 Intermediates 30 6.3 Reduced O2 Surrogates 33 6.4 Models of Thiolate-Ligated Nonheme Iron Enzymes with Other 3d Metals 37 7 Outlook and Future Work 40 References 42

1 Introduction

Nonheme iron and oxidases are a structurally and functionally diverse class of metalloenzymes that utilize dioxygen

(O2) to catalyze a wide range of oxidative transformations. Although the four-electron reduction of O2 is highly thermodynam- ically favorable, the ability of O2 to oxidize organic molecules is hampered by its remarkable kinetic stability. It has been hypothesized that the weak OdO s bond confers a significant thermodynamic driving force for O2 reactivity with organic molecules. However, the triplet ground state of O2 has four resonance structures, which provide a large resonance stabilization –1 energy (estimated to be –100 kcal mol ), strengthening the O2 p bond and rendering O2 inert towards spontaneous reactions with organic molecules.1

The kinetic inertness of O2 is circumvented in biological and synthetic by employing redox active metal ions that can bind and activate dioxygen to unleash its oxidative potency.2 The proposed mechanisms for many nonheme iron enzymes involves initial binding of dioxygen to generate an FeIII(superoxo) intermediate, which then proceeds to react with a or co-substrate, leading to a cascade of reactions involving other reduced oxygen species such as Fe((hydro)peroxo) (Fe(OO(H)), or high-valent Fe(oxo) (FeIV(O)) intermediates. One major class of dioxygen activating nonheme iron enzymes are those involving a co-substrate. The archetypical examples of this class are the a-ketoglutarate (a-KG) dependent nonheme iron enzymes, which undergo an oxidative decarboxylation of the IV a-KG co-substrate following binding of O2 to produce a high-valent Fe (O) species that carries out substrate oxidation. In contrast, a second class of nonheme iron oxygenases does not require a co-substrate.3 A major group of these enzymes function with sulfur- containing substrates, including isopenicillin N synthase (IPNS), the thiol dioxygenases, the sulfoxide synthases, and the persulfide dioxygenases. It is proposed that for each of these enzymes, their respective thiolate/persulfide substrates coordinate to the Fe centers through sulfur, promoting the binding and activation of O2. The focus of this chapter is the structures, functions, and proposed mechanisms for these oxidative, sulfur-ligated nonheme iron metalloenzymes, and the related synthetic, small-molecule transition metal complexes that serve as models for these systems.

Comprehensive Coordination Chemistry III https://doi.org/10.1016/B978-0-12-409547-2.14906-6 1 2 Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes

2 Isopenicillin N Synthase 2.1 Introduction Isopenicillin N is a member of an important class of molecules known as b-lactams.4 The broad importance of b-lactams stems from their ability to inhibit bacterial cell formation, making them powerful antibiotics used to treat a wide range of bacterial diseases.5 Despite displaying weak antibiotic activity,6 isopenicillin N is a precursor in the biosynthesis of an extensive list of potent antibiotics including other penicillins and cephalosporins.4 Isopenicillin N is produced in both bacterial and fungal organisms by a nonheme iron dependent , isopenicillin N synthase (IPNS), which uses a mononuclear iron and dioxygen to convert the linear tripeptide d-(L-a-aminoadipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N, a transformation that entails two oxidative cyclization reactions, each of which necessitates the cleavage of a strong CdH bond (Fig. 1). The significance of IPNS as a key entry point into a number of antibiotic biosynthetic pathways and as an Fe-dependent enzyme capable of catalyzing chemically challenging reactivity has impelled major research efforts into understanding the function and mechanism of this enzyme.

Fig. 1 Conversion of ACV to isopenicillin N by IPNS.

2.2 Structure and Function Prior to the first crystal structures of IPNS, the structures of IPNS with and without substrate were examined by sequence alignment – and mutagenesis studies,7 9 as well as spectroscopic techniques such as nuclear magnetic resonance (NMR),10,11 X-ray absorption (XAS),12,13 Mössbauer14,15 and electron paramagnetic resonance spectroscopies.15 Taken together, these studies found that the resting state of IPNS features a six coordinate Fe center ligated by two histidines, an aspartate, a glutamine, and two water molecules. It has been proposed that coordination of the ACV substrate leads to monodentate coordination via the thiolate donor. Nitric oxide 7 (NO) was employed as an O2 surrogate in the presence of the ACV substrate, leading to the formation of an iron nitrosyl ({FeNO} , S ¼ 3/2) species, which was characterized by EPR, Mössbauer, and optical spectroscopies.14 The first crystallographic evidence for the of IPNS from Aspergillus nidulans was obtained in 1995, with Mn substituted for Fe in the active site.16 The structure confirmed previous studies suggesting that the metal center was coordinated octahedrally by two histidine ligands, one aspartate, and one glutamine residue, and two water molecules. The crystal structure of IPNS containing both Fe and the ACV substrate revealed a 5-coordinate, trigonal bipyramidal metal ion, in which the ACV thiolate donor had displaced a Gln residue, and one water molecule was lost from the metal (Fig. 2).17 The two His and Asp ligands identified in the crystal represent a structural motif known as the 2-His-1-carboxylate triad, which is prevalent in a number of nonheme iron oxidases and oxygenases.18 The crystal structure of the {FeNO}7 form confirmed that a small diatomic molecule can bind to the Fe center in the presence of the ACV substrate.

Fig. 2 Crystal structures of IPNS-ACV complex (PDB: 1BK0) (left) and IPNS-ACV-NO complex (PDB: 1BLZ) (right). Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes 3

2.3 Mechanism Extensive work in the 1980s and 1990s provided the basis for understanding the mechanism of the four-electron oxidation of ACV catalyzed by IPNS19 and has been reviewed elsewhere.20 These elegant studies exploited easily derivatized tripeptide substrate analogues of ACV and the expanded substrate scope of IPNS to observe alternative distributions, and to prematurely stop the reaction cycle and intercept intermediates. The observed reactivity patterns led to the mechanistic picture shown in Scheme 1. It was hypothesized that conversion of isopenicillin N from ACV occurs via two sequential two electron oxidation/cyclization steps, b-lactam formation followed by thiazolidine formation. The b-lactam formation requires cleavage of the pro-S-Cys b CdH bond, and thiazolidine formation requires cleavage of the Val b CdH bond. It was proposed that these steps are mediated separately by an FeIII(superoxo) species and an FeIV(oxo) species, respectively, (Scheme 1) and it has been found that both CdH abstraction steps are partially rate-limiting.21 Although these studies relied upon organic product identification, they did provide a working mechanistic hypothesis for further analysis.

Scheme 1

Crystallographic methods were developed that involved exposure of anaerobic crystals to high O2 pressures, allowing for structural characterization of products following oxygenation in the solid state.22 In addition, the ability to crystallize IPNS in the presence of substrate/substrate analogues has allowed structural “snapshots” to be obtained of the reactions with various IPNS substrates. A number of these crystallographic studies expanded on the early reports from the 1980s and 1990s, and provided definitive structural evidence for the interaction between substrate analogues as well as oxidation products with the Fe center in the active site. The substrate analogue, d-(L-a-aminoadipoyl)-L-cysteinyl-L-S-methylcysteine (ACmC), which replaces D-valine with the thioether, S-methylcysteine, was used in order to prevent the second cyclization from occurring.22 Addition of ACmC to IPNS in the presence of Fe allowed for crystallographic characterization of the substrate-bound ferrous complex, confirming that this substrate analogue can bind to the Fe center as seen for the native substrate ACV. However, in this case, the thioether is bound in the open coordination site proposed for O2 binding. Addition of dioxygen at high pressure allowed for the formation of a monocyclic b-lactam product in which the thioether moiety was oxygenated to sulfoxide. Crystallographic characterization revealed that the substrate was bound to Fe via the thiolate donor and sulfoxide O-atom (Fig. 3). Thioether oxidation to sulfoxides by FeIV(oxo) species is well-established;23 therefore, these studies supported previous mechanisms suggesting that b-lactam formation occurs before thiazolidine formation, and that an FeIV(oxo) species is formed following the first ring closure step.

Fig. 3 (A) Reaction of IPNS with substrate analogues ACmC and (B) crystal structure of oxygenated product bound to IPNS (PDB: 1QJF). 4 Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes

O - CO - O CO2 2 H O AA N 2 N N HN H O AA S SH O AC-Me-cpG

- - - CO2 CO CO2 2 O H O O HN O N N O O O 2 O S OH S N AA HN NHis S H NHis NHis HN FeII FeIV AA FeIII AA NHis OH2 NHis OH2 NHis OH2 OAsp OAsp OAsp Scheme 2

The cyclopropyl-containing ACV analogue, d-(l-a-aminoadipoyl)-L-cysteinyl-b-methyl-D-cyclopropylglycine (AC-Me-cpG), 24 undergoes a ring-opening reaction in the presence of IPNS following addition of O2. The observation of ring-opened products was strongly indicative of the formation of a carbon-based radical. In an earlier report on this work, however, the proposed mechanism of product formation involved formation of an FedC bond prior to ring closure with the metal-bound thiolate. The crystal structure of AC-Me-cpG-bound IPNS indicated that the orientation of the cyclopropyl group strongly disfavors formation of an FedC bond, and instead is more consistent with a radical recombination mechanism in which an FeIV(O) generates the carbon- based radical via H-atom abstraction, which then attacks the metal-bound thiolate, as has been proposed with the native ACV substrate (Scheme 2).25 Early studies using other alkyl derivatives of the valine peptide of ACV revealed the possibility for alternate oxidation pathways following initial b-lactam formation. Oxidation of d-(L-a-aminoadipoyl)-L-cysteinyl-D-a-aminobutyrate (ACAb) in the presence of IPNS resulted in the formation of three oxidation products, in a 1: 7: 3 product ratio (Scheme 3). It was hypothesized that this ratio related to the ability of the FeIV(O) intermediate to abstract the CdH bond that would result in the formation each of these products.19 Crystallization of ACAb-bound to IPNS with and without a nitrosyl ligand revealed that binding of the substrate analogue was almost identical to that of ACV with exception of the aminobutyrate moiety, which was highly disordered over multiple positions. Modeling the relative energies of different conformers of the ACAb substrate revealed three distinct energy minima, which could be correlated with the observed product distribution. These studies suggest that the outcome of the second ring-closure step by IPNS is dictated by positioning of the alkyl chain within the active site.26

CO - CO - O - 2 2 CO - H O CO2 O O O 2 AA N 2 N H N HN N N O O S S H AA O H N N S SH AA H AA H O ACAb

1:7:3 Scheme 3

In order to interrupt IPNS prior to b-lactam formation, the substrate analogue, d-(L-a-aminoadipoyl)-L-cysteine D-a- hydroxyisovaleryl ester (ACOV), which replaces the amide linkage of ACV with an ester, was prepared.27 Addition of ACOVto IPNS allowed for crystallization of the substrate-bound complex, which revealed a similar structure to ACV-bound IPNS. However, addition of O2 resulted in the formation of a thiocarboxylate, in which the CdH bond that normally forms the b-lactam is oxidized to a carbonyl. This observation supports the hypothesis that an initial thioaldehyde intermediate is formed, which, in the absence of the amide moiety, reacts either directly with water or with the putative Fe(OOH) intermediate (Scheme 4). Computational studies aimed at understanding the observed reactivity suggested that nucleophilic attack of the thioaldehyde by an FeII(OOH) intermediate was energetically feasible.28 When a slightly altered analogue of ACOV, AmCOV, which contains a methyl group adjacent to the thiolate (Scheme 5), is used, a different reaction product is observed.29 In this case, a thiol-ene type product is formed, and the valine methyl group is hydroxylated. It was proposed that orientation of the valine residue away from the Fe center resulted in the unusual primary carbon-center hydroxylation. The fact that the radical recombines with the hydroxyl group rather than the thiolate was rationalized by the absence of the b-lactam, which could position the valine residue in close proximity to the thiolate for the native ACV substrate. Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes 5

Scheme 4

- CO - CO2 2 O O O AA O O NH O O N O2 NHis S H NHis S FeII AA FeII NHis OH2 NHis OH2 OAsp OAsp Scheme 5

One study used a substrate analogue that was a hybrid of ACOV and ACmC, d-l-a-aminoadipoyl-l-cysteine (1-(S)-carbox- y-2-thiomethyl)ethyl ester (ACOmC), which incorporates a thioether moiety in place of the valine and an ester in place of the amide normally found in ACV.30 X-ray crystallography reveals that ACOmC does bind to the Fe center of IPNS. Interestingly, addition of

O2 results in the formation of a new product in which the metal-bound thiolate has been oxygenated to a sulfenate, and the thiomethylcysteine unit has been hydroxylated (Fig. 4). It was proposed that this unusual reactivity is a result of O2 binding trans to His214, rather than Asp216, and that the methylcysteine hydroxylation step occurs first to generate an FeIV(O) intermediate that then S-oxygenates the metal-bound thiolate. An alternate mechanism in which S-oxygenation to form the sulfenate via an FeIII(superoxo) intermediate occurs prior to the CdH hydroxylation step was considered, but was deemed less likely. However, this alternative mechanism is in accordance with the proposed mechanism for cysteine dioxygenase (see Section 3.3). The extensive organic product analysis and crystallographic studies using ACV and substrate analogues strongly hint at the involvement of FeIII(superoxo) and FeIV(oxo) intermediates in the reactivity of IPNS. However, these studies failed to provide direct evidence for these Fe/oxygen intermediates, and as a result, little could be learned about their fundamental spectroscopic properties, III or their exact involvement in the mechanism of IPNS. Although the O2-bound, Fe (superoxo) intermediate in IPNS could not be characterized, a spectroscopic study employing EPR, circular dichroism (CD), and magnetic circular dichroism (MCD)

Fig. 4 Reaction of ACV substrate analogue ACOmV with IPNS (left) and crystal structure of IPNS-sulfenate complex (PDB: 2VBB). 6 Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes spectroscopies on the {FeNO}7 form of IPNS as an analogue of the FeIII(superoxo) intermediate was carried out.31 The spectro- III •− scopic data was accompanied by density functional theory (DFT) calculations, which indicated that the S ¼ 2 state of the Fe (O2 ) •− species should be lowest in energy. The calculations also suggested that the O2 unit should be properly oriented to accept the H-atom from the ACV substrate. The binding of O2 is calculated to be exergonic, which provides a possible rationale for why IPNS is 18 able to activate O2 in the absence of a co-substrate or cofactor, as is usually required for other nonheme iron enzymes. A number III •− of other computational studies have also been performed on IPNS, and suggest that mechanisms involving Fe (O2 ) and an – FeIV(O) intermediates are energetically feasible.28,32 34

In 2016, the first experimental evidence for the Fe/O2 intermediates responsible for each of the CdH cleaving steps in IPNS was obtained.35 Deuterated substrates were employed to enable the accumulation of intermediates prior to H-atom abstraction. A transient intermediate was observed in the IPNS reaction with the native ACV substrate, exhibiting UV-vis bands at 360 nm –1 –1 and 515 nm. Mössbauer spectroscopy on this species gave d ¼ 0.27 mm s and DEQ ¼ –0.44 mm s (Fig. 5). These parameters IV 36 are consistent with a high-spin nonheme Fe (O) species. Examination of the deuterated AC[d8-V], in which all eight valine H atoms are substituted with deuterium, showed that this intermediate precedes the second ring closing step in the formation of isopenicillin N. A kinetic isotope effect (KIE) 30 was measured for the decay of this intermediate with ACV. Together these findings pointed to the FeIV(O) assignment for this intermediate. A transient intermediate that precedes the formation of the designated FeIV(O) species was observed in low abundance by –1 −1 stopped-flow UV-vis (515, 630 nm) and Mössbauer (d ¼ 0.53 mm s , DEQ ¼ 1.02 mm s ) spectroscopies with the deuterated substrate A[d2-C]V. Preliminary variable-field Mössbauer data indicated an S ¼ 2orS ¼ 3 spin ground state. These spectral III •− 37 properties are similar to an Fe (O2 ) intermediate characterized in homoprotocatechuate-2,3-dioxygenase (HPCD), and together III •− the data point to an Fe (O2 ) species. The assignment of the two observed intermediates was supported by DFT calculations. Of the models tested for the FeIV(O) intermediate, an FeIV(O) (S ¼ 2) species complexed with the 2-His-1-carboxylate triad, the singly- cyclized b-lactam via the thiolate ligand, and one water molecule provided the best match for the observed Mössbauer parameters. For the proposed superoxo intermediate, models with the superoxide ligand coordinated in both a side-on and end-on fashion were III •− considered in the spin states S ¼ 1, 2, or 3, and only the calculated Fe (O2 ) structure with a quintet spin ground state provided a good match with the experimental spectroscopic data. Overall, these spectroscopic studies provide support for the mechanism proposed in Scheme 1. A recent computational study set out to address the origin of the selectivity for CdH activation over S-oxygenation in IPNS. III •− A hypothetical reaction pathway was calculated in which the Fe (O2 ) intermediate attacks the coordinated sulfur donor rather − than the Cys b CdH bond. It was found that the barrier for S-oxygenation is about 4 kcal mol 1 higher than that for CdH activation, due to an unfavorable steric interaction between the ACV valine alkyl groups and the Fe center on the S-oxygenation pathway. A calculated pathway in which the Val group is frozen lowers the barrier for S-oxygenation relative to H-atom abstrac- tion.38 However, the explanation involving sterics does not address why the significantly less bulky ACV substrate analogues ACG and ACA, which substitute the valine peptide with glycine and alanine, respectively, do not undergo any S-oxygenation reactivity.39

III0.0 A 4.0 0.0 0.2 0.0 1.0 D

0.0 0.0 1.0 C 0.2 [0–53mT] 0.0 D 1.0 δ

Absorption (%) Absorption (%) = 0.53 mm/s 0.0 |ΔEQ| = 1.02 mm/s 1.0 E 0.0 0.0 0.2 [0–13mT] 2.0 F

−4 −2 0 24 − − 4 2 0 24 Velocity (mm/s) Velocity (mm/s)

Fig. 5 (I) (A) Zero-field 4.2 K Mössbauer spectra of IPNSFe(II)ACV complex before adding O2. Zero-field 4.2 K Mössbauer spectra after adding O2 at 5 C and monitoring the reaction mixture at different time points trapped by a freeze-quench method: (B) 0.02 s, (D) 0.12 s, (E) 0.45 s, and (F) 90 s. Spectrum C is the difference spectrum between A and B. (II) Zero-field 4.2 K Mössbauer spectra of samples prepared by reacting IPNSFe(II)A[d2-C]V complex with O2 at 5 C and freeze quenching after 0.010 s. The top spectra show overlaid data of the experimental spectrum collected in the absence of a magnetic field (black vertical bars), in the presence of a 13 mT magnetic field (blue solid line), and in the presence of a 53 mT (red solid line). The middle spectrum shows the difference spectrum for the 0 field and 53 mT data, with the green line representing the best fit. The bottom spectrum shows the difference spectrum for the 0 field and 13 mT data. Reprinted with permission from reference Tamanaha, E.; Zhang, B.; Guo, Y.; Chang, W.-C.; Barr, E. W.; Xing, G.; St. Clair, J.; Ye, S.; Neese, F.; Bollinger, J. M., Jr.; Krebs, C. J. Am. Chem. Soc. 2016, 138, 8862–8874. Copyright 2016 American Chemical Society. Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes 7

3 Thiol Dioxygenases 3.1 Introduction The thiol dioxygenases are a class of nonheme iron enzymes that are responsible for converting thiol substrates into their corresponding sulfinic acids. The first discovered thiol dioxygenases were cysteine dioxygenase (CDO),40 which converts cysteine (Cys) to cysteine sulfinic acid (CSA), and dioxygenase, or 2-aminoethanethiol dioxygenase (ADO), which is responsible for converting cysteamine to hypotaurine (Scheme 6).40,41 Both of these enzymes are found in mammals and play critical roles in sulfur amino acid metabolism. The bacterial thiol dioxygenases, namely 3-mercaptopropionoic acid dioxygenase (3MDO) and mercaptosuccinate dioxygenase (MSDO), are also known (Scheme 6).42,43

Scheme 6

Cysteine dioxygenase represents the most well-studied thiol dioxygenase. It is found in mammals, bacteria, and plants, and is 44–46 known to generate CSA using O2 as the oxidant and the O-atom source. The formation of CSA represents the first step in the metabolism of Cys. Further breakdown of CSA eventually leads to the formation of pyruvate, sulfate, and taurine, which are important for various signaling pathways and metabolic functions.47,48 The cytotoxicity and neurotoxicity of high levels of cysteine are well-established,49 and evidence suggests that cysteine buildup is associated with a number of neurodegenerative and autoimmune diseases50 such as Parkinson’s disease,51 Alzheimer’s disease,52 rheumatoid arthritis,53 Hallervorden-Spatz – syndrome,54 as well as various cancers.55 57 Cysteine dioxygenases have also been shown to trigger N-terminal cysteine oxidation and initiates an N-degron pathway that controls responses to hypoxic conditions in plants and animals.45,46,58 −1 59–61 CDO is most prevalent in hepatic cells where cysteine levels range between 0.01 and 0.1 mmol L . The measured Km for − Cys consumption by CDO is 0.45 mmol L 1, significantly higher than cellular Cys levels,62 indicating that CDO is able to respond rapidly to changes in Cys concentrations in hepatocytes. CDO activity is regulated by polyubiquitination, which occurs at low levels of dietary Cys intake, and is inhibited when high levels of Cys are present,60 and as a result, CDO activity in liver tissue is tightly regulated by dietary cysteine levels. The same responses to dietary Cys intake are not observed for nonhepatic CDO; however, evidence suggests that CDO is able to alter intercellular cysteine levels, indicating that CDO plays a central role in controlling mammalian cysteine levels.49 Cysteamine, which is known to be produced by the catabolism of CoA,63,64 is oxidized by ADO to produce hypotaurine and eventually taurine, which has a wide range of physiological roles including regulating brain and cardiovascular function.65 ADO is found to be more widespread in various tissues compared to CDO.66 It is thus proposed that, like CDO, cysteamine dioxygenase plays an important role in maintaining proper human health. It has recently been shown that ADO’s primary role may be to act as an oxygen sensor in plants and animals. A study found that ADO regulates oxygen levels in response to hypoxia by initiating sulfination of an N-terminal, peptidic cysteine which triggers the eventual decomposition of G protein signaling regulators control GTPase activity.58

3.2 Cysteine Dioxygenase Structure and Function Early work on mouse (Mus musculus) and rat (Rattus norvegicus) CDO focused on sequence alignment studies and enzymatic activity assays, which indicated the importance of iron for activity,40,67,68 and that CDO is likely part of the cupin superfamily of enzymes, which includes a number of other nonheme iron enzymes such as homogentisate dioxygenase, quercetin dioxygenase, and the catechol dioxygenases.69 This superfamily is a diverse class of proteins typically characterized by two conserved peptide chain motifs

GX5HXH(X)3–6E(X)6G and G(X)5–7PXG(X)2H(X)3N (where X refers to amino acid residues that are not strictly conserved) that structurally leads to the b-barrel shape shared among the superfamily.69 The first crystal structure for CDO was obtained in 2006 from Mus musculus.70 This study provided important structural information about the active site of CDO, although it was solved with a catalytically inactive nickel ion,40,71 rather than iron, bound in the active site (Fig. 6). The most striking features from this structure revealed that the metal center in CDO is coordinated to three protein-derived His ligands (His86, His88, and His140, Fig. 6), forming a 3-His triad, which was an unusual departure from the 2-His-1-carboxylate (Asp/Glu) triad typically found in nonheme iron oxygenases.70 A Cys93-Tyr157 crosslink, which was unprecedented among cupin proteins, was also found within 4.2 Å of the Ni center. 8 Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes

Fig. 6 Crystal structure of Ni-substituted CDO from Mus musculus (PDB: 2ATF). Full protein structure shown on left and active site shown on right.

The first crystal structure of iron-constituted CDO was reported from the rat homolog, which has an identical sequence identity compared with mouse CDO.72 Like mouse CDO, rat CDO showed the metal center coordinated to a 3-His triad, in close proximity to a Cys–Tyr crosslink. Other conserved residues included Tyr58, Arg60, Ser153, and His155, which are all proposed to be important for substrate binding and/or catalysis. The Fe center in CDO appeared to be tetrahedrally coordinated, with water serving as the fourth ligand, an unprecedented geometry among nonheme iron enzymes. The oxidation state of the Fe could not be determined, but it was assumed to be in the ferric state. Attempts to isolate crystals of the substrate (Cys) bound form of rat CDO were unsuccessful. A separate study described extended X-ray absorption fine structure (EXAFS) measurements on rat CDO, and found II that the resting state was best modeled as having six N/O ligands, not four, consistent with an Fe (His)3(H2O)3 coordination sphere.71 Subsequent structural and Mössbauer studies on mammalian wild-type (wt) CDO, mutants, and bacterial CDO supported – that the resting ferrous state is indeed 6-coordinate.73 75 For years the structure of Cys-bound CDO remained controversial. An early EXAFS study suggested that the substrate Cys did not coordinate via the sulfur donor to the iron center.68 However, the first crystal structure of Cys-bound CDO (human), which exhibits 92% sequence homology with mouse and rat CDO, suggested that the Cys coordinates to the Fe via the S/N donors in a distorted trigonal bipyramidal geometry, with the 3-His triad comprising the rest of the coordination sphere.76 Interestingly, a later study reexamined these data and found that the electron density can be better modeled by rearranging the active site residues and replacing the Cys substrate with water, indicating that it is in fact not the Cys-bound structure “with” indicating that the original structure was not Cys-bound.77 This same study recrystallized substrate-bound CDO and found that Cys does in fact bind directly to Fe via N/S coordination, giving a five-coordinate structure and an FedS distance of 2.35 Å at pH 8 (Fig. 7). The direct coordination of cysteine was also supported by a number of other structural,77,78 Mössbauer,73 magnetic circular dichroism,79,80 and computa- tional studies.79,80 The Mössbauer data on Cys-bound CDO showed broad features consistent with the presence of two quadrupole doublets.73,81 One possible explanation was that incomplete Cys–Tyr crosslink formation gives two slightly different iron environ- ments. However, Mössbauer studies on the CDO mutant C93G, which is unable to form the crosslink, also revealed the presence of two quadrupole doublets, ruling out this hypothesis. The possibility that Cys-bound CDO exists as a mixture of 5-coordinate and 6-coordinate iron, with water serving as the possible sixth ligand, was proposed. This idea was supported by studies on a series of synthetic model complexes (see Section 6.1).82

Fig. 7 Crystal structure of Cys-bound human CDO (PDB: 4IEV). Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes 9

The unusual Cys93-Tyr157 crosslink in CDO is also found in the copper-containing metalloprotein galactose oxidase.83 Unlike galactose oxidase, however, CDO exhibits catalytic activity in both the presence and absence of this crosslink, as seen in studies involving the immature form of the protein (i.e., prior to crosslink formation), and in mutant forms of the protein in which Cys93 is substituted. It has been demonstrated that the crosslink is a post-translational modification84 that occurs only in the presence of protein-bound iron, dioxygen, and the substrate, cysteine, and the enzyme requires 800 turnovers before reaching 50% matura- tion.85 These data suggest that the crosslink forms over the course of catalysis, though relatively inefficiently.85 An early proposed mechanism implicated formation of an FeIII(superoxo) intermediate, which oxidizes Tyr157 prior to insertion of the Cys93 sulfur atom.85 Efforts were made to examine the biogenesis of the Cys–Tyr crosslink by engineering modified CDO variants using fluorine and chlorine substituted tyrosine analogues. Surprisingly, even a mutant containing 3,5-di-fluoro-substituted tyrosine (F2-Tyr157) CDO generated the Cys–Tyr crosslink, suggesting that a potent oxidant capable of cleaving a strong CdF bond is formed during 74 crosslink formation (Fig. 8). The crystal structures of the Fe–nitrosyl adducts of F2-Tyr157 CDO before and after crosslink 86 formation were obtained, and revealed a strong interaction between F2-Tyr157 and the Cys carboxylate group prior to crosslink formation, as well as an interaction between the nitrosyl O atom and Cys93. These data led the authors to conclude that an Fe(superoxo) species may abstract an H-atom from Cys93 to generate a thiyl radical, which then inserts into the Tyr to form the crosslink. It has been noted, however, that crosslink formation in vivo may occur via different mechanisms than those occurring in vitro.84

Fig. 8 Crystal structures of engineered CDO mutant with F2-Tyr157 complexed with Cys prior to (left) and after (right) crosslink formation (PDB: 6BPS (left) and 6BPV (right)).

The exact role of the crosslink has been subject to intense debate. Initial studies found that the presence of the crosslink increases with higher concentrations of Cys, and enhances the catalytic efficiency of the enzyme.85 These results suggest that the crosslink may serve a regulatory role in response to cellular Cys levels. From a mechanistic perspective, the crosslink is known not to be essential for catalysis in mammalian CDOs. Furthermore, bacterial CDOs contain a conserved Gly in place of Cys93 and still exhibit high activity.87 Comparisons between wt-CDO and C93G-CDO revealed that turnover occurs with similar levels of efficiency, although at different optimum pH levels,88 and it was proposed that the crosslink serves to prevent competing reactions at Cys93. Other proposals have suggested that the crosslink serves a structural function, promoting optimal binding of Fe, substrate, or properly – orienting intermediates formed during catalysis.89 91 In line with these proposals, it has been found that the crosslink may prevent oxidative uncoupling during CSA production.92 CDO exhibits a high degree of substrate specificity (Scheme 7). There are numerous reports demonstrating the inability of CDO to oxygenate other sulfhydryl containing molecules, such as cysteamine, 3-mercaptopropionoic acid, homocysteine, 2-aminothiophenol, as well as the selenium analogue of Cys, selenocysteine (sec).68,71,80,93,94 One report suggested that CDO 94 can undergo some activity with homocysteine and L-penicillamine, albeit inefficiently. Interestingly, it has been shown by 73 Mössbauer spectroscopy that homocysteine can bind to CDO in a similar fashion to Cys, yet does not react with O2. Spectroscopic studies have demonstrated that selenocysteine (sec) binds to both FeII and FeIII forms of CDO, and sec-bound FeII CDO forms an II adduct with nitric oxide. However, sec-bound Fe CDO exhibits no spectral changes upon addition of O2, and there is no evidence for the formation of oxidized organic products.79,80 The origin of CDO’s substrate specificity was discussed in a combined enzyme/ model study, where it was shown by Mössbauer spectroscopy that the non-native substrate 2-aminothiophenol does not chelate properly to FeII CDO and as a result, no S-oxygenation is observed.82 In contrast, chelation of 2-aminothiophenolate to FeII model complexes leads to efficient S-oxygenation to give the sulfinate product. It was hypothesized that proper S/N substrate chelation to FeII-CDO is likely required for successful S-oxygenation, and this requirement may be a significant contributor to CDO’s substrate specificity (see Section 6.1). In addition to oxygenating free cysteine, recent evidence has suggested that cysteine dioxygenation can occur in N-terminal, protein-derived cysteinyl residues mediated by plant cysteine oxidases. The occurrence of 10 Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes

Scheme 7

1 O2-derived cysteinyl dioxygenation was supported by H NMR studies as well as mass spectrometry using isotopically labeled O2 and water sources.45,46

3.3 Cysteine Dioxygenase Mechanism Early mechanistic proposals for CDO were aligned with the assumption that Cys does not bind directly to the Fe center, which was later shown to be incorrect. These proposals have been summarized elsewhere, but will not be discussed here.68 Most current III mechanistic hypotheses include an initial O2 binding step to form an Fe (superoxo) intermediate, although definitive character- ization of such a species has yet to be obtained (Scheme 8).

Nitric oxide (NO) is commonly used as a surrogate and spectroscopic probe for O2 binding in iron enzymes. Some data on CDO 79,95 II suggest that Cys must bind first to the Fe center in order to allow for O2 binding. Addition of NO to Cys-bound Fe -CDO results in the formation of an {FeNO}7 species, which was observed by EPR spectroscopy. Unlike the vast majority of {FeNO}7 species in nonheme iron enzymes, which exhibit an S ¼ 3/2 ground state, the {FeNO}7 species of CDO exhibits a low-spin, S ¼ 1/2 ground state. Density functional theory calculations suggested that the ls-{FeNO}7 should be a six-coordinate iron species with NO bound in the sixth site. It was hypothesized that the O2 may bind in a similar fashion, occupying the sixth site through an “end-on” bonding mode. The unusual spin state of the {FeNO}7 form may arise from the rather different coordination environment of Cys- bound CDO (sulfur ligation, 3-His triad) compared to other mononuclear nonheme Fe enzymes. However, it is unlikely that the presence of the thiolate donor alone is responsible, because the{FeNO}7 form of IPNS, which is also thiolate-ligated, exhibits the more typical S ¼ 3/2 ground state.31 III •− Attempts to chemically produce the Fe (superoxo) intermediate involved the addition of O2 to the ferric form of Cys-bound CDO, which was proposed to be an FeIII(OH) species from EPR, MCD, and resonance Raman measurements.96 The (Cys)FeIII(OH) •− III form is catalytically inactive; however, addition of O2 results in the rapid decay of (Cys)Fe (OH) CDO (lmax ¼ 630 nm) and formation of a new intermediate with l ¼ 485 and 565 nm as seen by UV-vis spectroscopy (Fig. 9).97 This new species has a lifetime of minutes, and its decay was kinetically matched with the formation of CSA, although CSA production was 200 times slower than III during native catalysis with O2. Samples of the Fe enzyme after treatment with superoxide were restored to normal CDO catalytic •− activity. EPR characterization of the O2 intermediate indicated that it exhibited an integer spin, S ¼ 3 ground state. The authors hypothesized that this intermediate was an FeIII(superoxo). Whether this species lies along the catalytic cycle of CDO is unclear, given its high stability and low rate of CSA formation. This intermediate may be entirely off-pathway, or it may be the result of a slightly altered local environment. Further studies are needed to confirm the identity of this transient intermediate. Though most mechanisms support formation of an FeIII(superoxo) intermediate, they diverge after this step. These mechanisms primarily differ in the redox role played by the Fe center in the oxygenation of the thiolate donor. One proposed mechanism suggests that Fe oxidizes the thiolate via two, sequential mono-oxygenation steps involving initial O-atom transfer from FeIII(su- peroxo) to generate an FeIV(O)(sulfenate) intermediate, followed by a second O-atom transfer from the ferryl to generate a sulfinate-bound, ferrous product (Scheme 8, Mechanism I). This mechanism shares some commonalities with the proposed mechanism for a-KG-dependent nonheme iron enzymes such as taurine dioxygenase.18 Other proposals have suggested that dioxygenation occurs directly on the sulfur donor, in analogy to the proposed mechanisms for the S-oxygenation of Ni–thiolate complexes (Scheme 8, Mechanism II).98 Credence for the direct S-attack mechanism was provided by the crystallographic characterization of an Fe(persulfenate) species in Cys-bound CDO (Fig. 10), which was generated by soaking crystals of CDO with excess substrate in air.99 The Cys S atom and the proximal oxygen of the persulfenate moiety were bound to Fe, forming a three-membered ring structure. Because of the stability of Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes 11

Scheme 8

the crystals, it was questioned whether this structure represented a catalytic intermediate or an off-pathway species. The authors later hypothesized that a structurally similar intermediate (e.g., coordinated persulfenic acid) may represent the functioning, catalytically relevant intermediate.77 The crystalline, persulfenate form of CDO was incapable of generating the expected sulfinate product, suggesting that this species may be off-pathway.100 Computational studies (DFT and QM/MM)101,102 describe a model that involves initial formation of an S ¼ 0FeIII(superoxo) 5 intermediate, which rapidly undergoes spin state crossing to become the S ¼ 2FeIII(superoxo) species ( A), which then attacks the 5 S atom of a coordinated Cys via the distal oxygen atom, generating an S ¼ 2, four-membered FedOdOdS ring structure ( B) (Figure 11). Sulfur attack is calculated to be the rate determining step. The FedOdOdS intermediate then undergoes homolytic 5 OdO bond cleavage to produce an FeIV(O)(sulfenate) species ( C). Following S]O bond rotation, the Fe(oxo) species may then perform an O-atom transfer to the sulfenate to generate the ferrous-bound sulfinate product, which can then be released to regenerate the resting state FeII complex. Calculations on an alternate mechanism (Mechanism II) involving a persulfenate intermediate found that this pathway was significantly higher in energy, suggesting that it might not be a viable mechanism. Other DFT calculations were later performed on these hypothetical intermediates by using computational methods that accurately modeled spectroscopic data for the CDO {FeNO}7 species. These calculations indicated that the mechanism in Fig. 11 was viable and suggested that the S ¼ 2FeIII(superoxo) intermediate is the lowest energy spin state. A significant amount of spin density was II •− • III • also calculated to lie on the S atom, suggesting partial thiyl radical character in the superoxo (Fe (O2 )( SR)) and ferryl ((Fe (O ) • ( SR)) intermediates.79 12 Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes

•O (OH2) OH O H2 H2 H − N − N •− − 2 N O2C N HIS IrIV O2C N HIS O2 O2C N HIS FeII FeIII FeIII S NHIS S NHIS S NHIS NHIS NHIS NHIS

-CSA

0.85 0.55 (A) (B) 1.0 (C)

0.67 0.43 0.8 630 0.6 630 0.49 0.31 565 0.4 565 485 0.31 0.19 0.2 Absorbance

Normalized Concentration 0.0 0.13 0.07 0.15 0.00 −0.05 −0.05 −0.15

360 468 576 684 792 900 360 480 600 720 840 960 Residuals 0246810 λ/nm λ/nm time/min

III IV •− III Fig. 9 Proposed formation of Fe (superoxo) in CDO from reaction of ferric CDO (generated from oxidation with Ir ) with O2 . (A) UV-vis spectra of (cys)Fe (OH) CDO •− (dashed line, white circle) and after formation of O2 generated in situ from xanthine oxidase, with dark gray, light gray, and white circles indicating spectra collected III •− at 0.5, 1.0, and 2 s, respectively. (B) UV-vis spectra of (cys)Fe (OH) CDO (dashed line, white circle) and following addition of preformed solutions of O2 , with dark gray, light gray, and white squares indicating spectra collected at 0.5, 22, and 600 s, respectively. (C) Plot of concentration of CSA (gray circles) versus time and normalized decay of proposed FeIII(superoxo) species (black circles) versus time. Fits for formation of CSA and decay of FeIII(superoxo) species are shown as dashed and solid lines, respectively. Reprinted with permission reference Crawford, J. A.; Li, W.; Pierce, B. S. Biochemistry 2011, 50, 10241–10253. Copyright 2011 American Chemical Society.

Fig. 10 Crystal structure of the CDO Fe(persulfenate) species (PDB: 3ELN).

Experimental evidence for the mechanism of action of CDO has been hampered by difficulties in trapping catalytically relevant intermediates. However, other methods have provided some information regarding the details of the S-oxygenation mechanism. One study showed that wt-CDO catalysis exhibited a solvent KIE, suggesting that a proton transfer step is important in Cys oxygenation.103 It was proposed that the Tyr moiety may serve in an acid-base role during catalysis.88 However, it is unlikely that the Tyr plays a redox role (e.g., serving as an H-atom donor) since addition of radical scavengers does not effect catalytic efficiency, and no spectroscopic evidence for a tyrosyl radical has been observed.95 In 2016, the first report appeared of an intermediate trapped during native CDO catalysis (Fig. 12).81 This intermediate was stable for only 20 ms, and exhibited two bands at 500 nm and 640 nm by UV-vis spectroscopy. It was shown to be on the catalytic pathway. The absorption spectra for possible intermediates were calculated using DFT and ab initio methods, and the computed spectrum for the proposed FedOdOdS intermediate matched well with the spectrum seen for the 20 ms intermediate. However, the calculations also indicated that the spectrum could be III •− consistent with an Fe (O2 ) intermediate. More data are needed to definitively characterize this intriguing CDO Fe/O2 intermediate. Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes 13

Fig. 11 Reaction energy landscape for singlet, triplet, and quintet spin states for CDO calculated by DFT. Reprinted with permission from reference Aluri, S.; de Visser, S. P. J. Am. Chem. Soc. 2007, 129, 14846–14847. Copyright 2007 American Chemical Society.

3.4 ADO/3MDO/MSDO—The Other Thiol Dioxygenases Compared to CDO, significantly less is known about the other thiol dioxygenases. One major setback in understanding the other thiol dioxygenases is a lack of structural data for these proteins in the presence of their native substrates. The mammalian enzyme cysteamine dioxygenase (ADO) converts 2-amino-ethanethiol (cysteamine) to hypotaurine by oxida- tion of the thiol group. This reaction is analogous to Cys oxidation by CDO, and is critical for the production of taurine, a biomolecule that has many important roles and is widespread in various tissues.65 The participation of ADO in hypotaurine production was first described in the 1960s, where it was shown to convert cysteamine to hypotaurine.104 However, since the original work on ADO over 40 years ago, there is still relatively little known about the mechanism or active site structure, in part because there are still no X-ray structures available for this protein. Early studies found that ADO, like CDO, exhibits substrate specificity, and other thiols such as cysteine, glutathione, coenzyme A, or lipoic acid were neither substrates nor inhibitors of ADO.105 Some evidence suggested that ADO can S-oxygenate 2-mercaptoethanol, homocysteine, N-acetylcysteamine, and 3-mercaptopropionic acid.106 The characterization of human ADO was first reported in 2007,66 and in this study the previously observed substrate specificity was reiterated, as well as the requirement of iron for proper enzymatic function. This study also found that, in contrast to previous reports, exogenous reductants were unnecessary for catalysis. It was hypothesized that ADO may contain the unusual Cys–Tyr crosslink seen in CDO. Efforts to test this hypothesis included genetically encoding the unnatural amino acid 3,5-di-fluoro-tyrosine (F2-Tyr222) in ADO, as done in similar studies with CDO (see Section 3.2).107 Mass spectral analysis revealed that one of the CdF bonds is cleaved and a new CdS bond is formed with Cys220, confirming crosslink formation. The presence of the crosslink enhances catalytic activity of ADO, and thus may serve a similar function to that in CDO. The cleavage of a CdF bond suggested that a potent oxidant may be formed during crosslink biogenesis. Taken together, these results highlight that ADO might have similar structure and function compared to CDO, but clearly more work is needed to define the mechanism of ADO. It has recently been shown that ADO can oxygenate N-terminal cysteine during hypoxia to produce the protein-bound sulfinic acid, which was verified with mass spectrometry and isotope labeling studies.58 Competition experiments revealed that dioxygenation of the peptide cysteinyl residue is inhibited only at concentrations greater than 37 mM or 13 mM for free cysteamine or cysteine, respectively. It has thus been suggested that ADO may primarily functions as part of the N-degron pathway in mammals. The mechanism of the dioxygenation of protein-bound cysteine and how it compares to the oxygenation of free cysteine seen in CDO is not understood. 14 Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes

Fig. 12 (A) UV-vis absorption spectra showing decay of Fe/O2 intermediate (blue line) over 20 ms. (B) Plot of absorbance versus time for decay of Fe/O2 intermediate. (C) UV-vis spectra of Fe/O2 intermediate at 500 mM(red) and 240 mM(black) protein concentrations. (D) Plot of absorbance of Fe/O2 intermediate versus O2 concentration. (E) Plot of decay rate of Fe/O2 intermediate versus O2 concentration. (F) Plot of amount of CSA produced versus time of reaction of CDO + Cys + O2 before quenching. (G) Mössbauer spectra of CDO in absence of Cys (top), after adding Cys (second panel), after adding O2 for 10 ms (third panel), and after adding O2 for 20 ms (bottom). Reproduced from reference Tchesnokov, E. P.; Faponle, A. S.; Davies, C. G.; Quesne, M. G.; Turner, R.; Fellner, M.; Souness, R. J.; Wilbanks, S. M.; de Visser, S. P.; Jameson, G. N. L. Chem. Commun. 2016, 52, 8814–8817. with permission from The Royal Society of Chemistry.

The alkylthiol 3-mercaptopropionic acid (3-MPA) is converted to 3-sulfinopropionic acid (3-SPA) in the bacterium Variovorax paradoxus, with a high specificity for 3-MPA over Cys.108 It was hypothesized that the enzyme responsible might be a 3-mercaptopropionic acid dioxygenase (3MDO). A structural study on bacterial thiol dioxygenases found that these enzymes could be categorized into two structural classes, “Arg-type” and “Gln-type.” The “Arg-type” are cysteine dioxygenases and contain an Arg residue equivalent to Arg60 in mammalian CDO. The “Gln-type” contain a Gln residue in place of Arg and were classified as 3MDOs, rather than CDOs (Fig. 13).75 The 3MDO from Azotobacter vinelandii is not as selective as CDO towards its native substrate 3-MPA, and is able to catalyze S-oxygenation of Cys and cysteamine, albeit less efficiently than 3-MPA. Studies reacting NO(g) (an

O2 surrogate) with 3MDO showed that NO would only bind to the enzyme in the presence of substrate, as seen for CDO, suggesting that substrate binding most likely precedes O2 binding. In contrast to the nitrosyl adduct of CDO, the nitrosyl adduct of 3MDO was characterized as having an S ¼ 3/2 ground state. Similar S ¼ 3/2 species were formed in the presence of cysteine, cysteamine, or ethanethiol, suggesting that all of these compounds may bind to Fe only through the sulfur atom (Fig. 13).109 Mössbauer studies on 3MDO from Pseudomonas aeruginosa suggests binding of 3-MPA via the sulfur donor.42 An Arg residue is located further from the Fe center than Arg60 in CDO, and is positioned to form a salt bridge with the carboxylate group of 3-MPA, which could induce S coordination of 3-MPA.110 Recent substrate docking studies suggested that 3MDO from Pseudomonas aeruginosa could be modeled to bind 3-MPA via the sulfur atom and weakly with the carboxylate O-atom.110 Mechanistic information for 3MDO is relatively scarce, but 3MDO activity appears to be highly sensitive to pH, and likely requires the nearby tyrosine residue for H-bonding interactions or acid/base reactivity.111,112 Interestingly, unlike CDO, 3MDOs do not have a Tyr-Cys crosslink, containing a Gly in place of the cross-linking Cys. When 3MDO was mutated with Cys in place of Gly, evidence for an artificial crosslink formation was observed, but activity was significantly diminished.111 Further structural and mechanistic studies are needed to elucidate the mechanism of 3MDO. Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes 15

Fig. 13 Crystal structure of 3MDO from Pseudomonas aeruginosa (left). Scheme showing formation of {FeNO}7 in 3MDO from Azotobacter vinelandii (right).

A proteomics study in 2014 identified a possible thiol dioxygenase in Variovorax paradoxus that may be responsible for the degradation of mercaptosuccinate.113 This enzyme was found to exhibit high sequence similarity to CDO. Further studies demonstrated that Variovorax paradoxus converts mercaptosuccinate (ms) to succinate and sulfite, likely via the intermediacy of sulfinosuccinate. This enzyme requires Fe for proper function and is highly selective towards ms, suggesting that it is a true mercaptosuccinate dioxygenase (MSDO).43 Mutagenesis studies were performed to identify key residues involved in catalysis.114 The MSDO enzyme contains three conserved His residues like CDO, which upon substitution abolished reactivity. An Arg residue, analogous to Arg60 in CDO, is present and is essential for catalysis, suggesting that it may serve to help bind the succinate, possibly via a salt-bridge with the substrate carboxylate group. Mutation of a Gln residue positioned near the active site in 3MDO from Pseudomonas aeruginosa to an Arg leads to MSDO activity.110 It was proposed that this mutation allows for the ms carboxylates to form two salt bridges that favor proper substrate binding/orientation. Though no crosslink is present, a Tyr analogous to Tyr157 in CDO is present in MSDO. However, unlike in CDO, mutation to phenylalanine does not eliminate activity. This observation is a significant departure from that seen for the other thiol dioxygenases. Further structural and mechanistic studies are needed for this newest member of the thiol dioxygenase family.

4 Sulfoxide Synthases 4.1 Introduction Ovothiol A and ergothioneine are both sulfur containing histidine derivatives that are thought to be important biomolecules due to their antioxidant properties. Ovothiol A is found in a number of marine organisms and was first isolated from sea urchin eggs.115 It has a sulfhydryl pKa of 1.4 and a much more positive redox potential than other sulfur-based antioxidants such as glutathione (GSH). It is thought that ovothiol may scavenge peroxides or serve as a chemical signaling agent; however, its exact biological role is unknown.116 It has been hypothesized that the unique properties of ovothiol derivatives may lead to potential use in therapies targeting neurodegenerative diseases117 as well as tumor growth.118 Ergothioneine is a related sulfur-containing histidine derivative that exists primarily in its thione form. Like Ovothiol A, the redox potential is relatively high compared to typical thiol antioxidants,119 and as a result is much more stable to oxidation. Though ergothioneine is produced by fungi and bacteria, it is found in both plants and mammals and is obtained through diet. It has been proposed that ergothioneine may function as an antioxidant and metal chelator and may protect against neurodegenerative and cardiovascular diseases.119,120 In 2010, a series of five genes egtA, egtB, egtC, egtD, and egtE were shown to be responsible for the conversion of histidine to ergothioneine in Mycobacterium smegmatis. The EgtB gene produces a protein with a nonheme iron cofactor that is involved in an unusual CdS bond formation and sulfoxidation reaction between g-L-glutamyl-L-cysteine (g-GC) and Na-trimethyl histidine (TMH) (also known as hercynine).121 The enzyme OvoA was discovered around the same time as EgtB, and is responsible for a similar transformation in the biosynthesis of ovothiol A. Like EgtB, OvoA is a nonheme iron dependent enzyme that catalyzes a CdS bond formation and sulfoxidation reaction; however, OvoA was shown to use histidine and cysteine as its native substrates and performs the CdS bond formation at the imidazole C5 position rather than in the C2 position as seen for EgtB (Fig. 14).122 A related enzyme Etg1 was later discovered in Neurospora crassa, which performed a similar sulfoxide synthase reaction using cysteine instead of g-GC.123 Further phylogenetic analyses have found that EgtB-like sulfoxide synthases are widespread in a number of different organisms and can be classified into five different types, each with different structural motifs and substrate require- ments.124 Likewise, OvoA was also found to have over 80 homologues in protobacteria, microalgae, and marine organisms.116,123 The CdS bond formation and sulfoxidation reaction performed by these sulfoxide synthases enzymes are mechanistically complicated and unprecedented among nonheme iron enzymes. This mechanistic uncertainty, as well as the ubiquity and potential significance of ovothiol/ergothioneine biosynthesis makes the sulfoxide synthases an important emerging class of nonheme iron enzymes for structural and mechanistic examination. 16 Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes

Fig. 14 Reactions of EgtB and OvoA in the biosynthesis of ergothioneine and ovothiol A.

4.2 Structure and Function Following the discovery that OvoA is an Fe dependent nonheme iron enzyme, it was hypothesized that OvoA contains a 2-His-1-carboxylate type coordination geometry based on the protein sequence122; however, it was noted that the proposed metal-binding residues were unusually close in proximity in the sequence compared to most 2-His-1-carboxylate enzymes. Interestingly, mutation of E176, which was hypothesized to be the possible carboxylate donor, to alanine, significantly diminished sulfoxide synthase activity.125 No crystal structure for OvoA has been obtained. However, the crystal structure of EgtB from Mycobacterium thermoresistibile (MthEgtB) bound to Fe in the absence of substrates, and EgtB reconstituted with Mn in the presence of g-GC and TMH, were both obtained in 2015.126 The crystal structure of Fe-bound EgtB in the absence of substrates revealed that Fe is coordinated to three facially arranged His residues, much like that seen for CDO. Additionally, Tyr377 was shown to be positioned near the Fe center and was proposed to play a role during catalysis. Attempts to crystallize EgtB with Fe in the presence of g-GC and TMH were unsuccessful. However, addition of g-GC to the enzyme under aerobic conditions resulted in the formation of a UV-vis absorption feature at 565 nm, which could be attributed to an S!Fe charge-transfer transition, indicating that the thiolate likely binds directly to the Fe center. Further evidence of direct coordination of g-GC came from the structure of the catalytically inactive Mn-reconstituted EgtB. The structure shows the six-coordinate Mn coordinated by g-GC through sulfur, which also forms a salt bridge with Arg87 and Arg90, the TMH imidazole, one water molecule, and the 3-His triad (Fig. 15). Recent crystal structures of EgtB from Chloracidobacterium thermophilum (CthEgtB) revealed an entirely new structural motif for the sulfoxide synthases.124 The crystal structure of Fe-bound CthEgtB with and without TMH were both determined, and revealed that like MthEgtB, the Fe center is coordinated by the 3-His triad. However, CthEgtB differs in its overall protein structure and sequence from MthEgtB. In addition, CthEgtB was found to contain two tyrosine residues, Tyr93 and Tyr94, in close proximity to the Fe active site, rather than the single Tyr residue, Tyr377, seen in MthEgtB. In addition, CthEgtB uses cysteine rather than g-GC as its native substrate, and the ability to select for this substrate may come from minor changes in structure. MthEgtB is highly substrate specific and does not accept Cys or histidine as a substrate,127 and likewise, CthEtgB does not oxidize g-GC.

Fig. 15 Crystal structure of Mn-substituted MthEgtB (PDB: 4X8D). Overall dimeric structure (left); active site (right). Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes 17

Fig. 16 Reactions of mthEgtB and OvoA with native and non-native substrates.

OvoA selectively utilizes L-cysteine as the sulfur source, with no activity observed with D-cysteine, N-acetyl-L-cysteine, glutathi- 128 127,128 one, or thiophosphate. Conflicting results have been reported for the use of g-glutamyl-L-cysteine (g-GC) as a substrate. The specificity for imidazole substrates is much lower, with reactivity being observed for histamine, L-histidinamide, D-histidine, 2-fluoro-L-histidine, and 4-methyl imidazole. The apparent reactivity of 2-fluoro-L-histidine is particularly intriguing because it is significantly less electron rich than L-histidine, suggesting that the redox potential of the imidazole substrate may not play a large role in the mechanism of action of OvoA. The regioselectivity of the substitution of imidazole at C2 or C5 is dependent on the degree of methyl substitution on the histidine amine.128 When the native EgtB substrate, hercynine, is added to OvoA, substitution of the C2 position is observed as seen in EgtB (Fig. 16). However, the major product in this reaction was cysteine sulfinate, suggesting that OvoA can serve as a cysteine dioxygenase, and the nature of the substrate can steer the selectivity of OvoA toward CDO-type or sulfoxide synthase activity.125 It was also noted that a small amount (10%) of cysteine sulfinate is observed even in native catalysis with histidine. However, when cysteine is added to OvoA in the absence of an imidazole substrate, only the disulfide product, cystine, is observed. The formation of cystine is enzymatic; however, it is unclear if an initial S-oxygenated sulfenate species is formed, which then could lead to disulfide formation.129

4.3 Mechanism The differences in stereoselectivity and substrate scope for EgtB and OvoA as well as the observation of both CDO-type reactivity and sulfoxide synthase reactivity have important implications for their mechanisms. The products from these enzymes point to an obvious mechanistic question: does CdS bond formation or sulfoxidation occur first? Two possible mechanistic pathways are shown in Scheme 9. Pathway I involves initial CdS bond formation followed by S-oxygenation, and Pathway II involves initial S-oxygenation, similar to that proposed for CDO, followed by CdS formation. Many of the mechanistic studies on these enzymes have been performed with this question in mind. EgtB can be altered to exhibit CDO-like dioxygenation reactivity.130 Mutation of Tyr377 in MthEgtB to phenylalanine results in nearly identical values of Km, suggesting that substrate binding is not significantly altered. However, the predominant product observed is the S-dioxygenated product, with only a small amount of the sulfoxide synthase product being produced. The observation of CDO-like reactivity suggests that MthEgtB and CDO may exhibit a common intermediate followed by a branching point that dictates the two reactivity patterns. No KIE was seen upon deuteration of TMH, suggesting that TMH C–H cleavage is not involved in the rate determining step. These mutagenesis study suggests that proton movement involving the Tyr residue may play a critical role in the branching point that determines product selectivity. The observation of two Tyr residues (Tyr93 and Tyr94) in close proximity to the active site of CthEgtB indicates that the role of Tyr in catalysis may be different for the different enzymes.124 Mutagenesis studies of CthEgtB show that when Tyr93 is substituted with Phe, CSA is the major product, but sulfoxide synthase activity is still observed. Substitution of Tyr94 with Phe significantly diminishes Cys consumption, suggesting that Tyr94 may be involved in O2 binding/activation. OvoA exhibits both CDO and sulfoxide synthase activity, and an isotope sensitive branching method131 showed that no kinetic isotopic effect was observed with a fully deuterated histidine substrate. A small solvent KIE of 1.29 is observed when D2O was used as the solvent, suggesting that protonation steps may be involved in the mechanism.122,128 Given that no crystal structure for OvoA has been obtained, it is difficult to determine specifically which residues are involved in catalysis. However, sequence alignment suggests that Tyr417 in OvoA may be the equivalent of Tyr377 in EgtB, which is known to play a role in sulfoxide synthase activity. 18 Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes

Scheme 9

The Tyr417 has been substituted with artificial amino acid tyrosine analogues in order to examine its role in sulfoxide synthase activity.131,132 A 2-methylthio-substituted tyrosine analogue (MtTyr) (similar to the Cys–Tyr crosslink in CDO)131 gave a slight increase in the ratio of CSA:native sulfoxide from 1:9 to 3:7. This observation suggests Tyr417 is involved in the branch point between CDO-like and sulfoxide synthase activity. It must be noted that MtTyr alters both the redox potential and pKa relative to tyrosine. Incorporation of another tyrosine analogue, MeOTyr, in which a methoxy group is substituted ortho- to the phenol moiety, provided a second mutant in which the pKa of the unnatural MeOTyr is much closer to that of Tyr, but the redox potential is much lower than that of Tyr.132 More CSA is produced with MeOTyr-OvoA (26%) as compared to native OvoA (10%). An inverse KIE of 0.86 was observed for MeOTyr-OvoA with a fully deuterated His substrate, compared to a KIE of 1.0 for native OvoA. These KIEs suggest that OMe substitution at Tyr417 changes the rate-limiting step for OvoA catalysis. These results together with other findings led the authors to favor Pathway I. However, if the branching point for CDO-like and sulfoxide synthase activity occurs following III •− formation of Fe (O2 ), it would be expected that incorporation of MeOTyr, would make the H-atom abstraction more favorable relative to the CDO-like pathway, which should lead to the production of less CSA relative to native catalysis. The observation of more CSA with MeOTyr-OvoA contradicts this hypothesis. Computational studies have been carried out to examine the mechanism of the sulfoxide synthases. Several mechanisms were proposed, and these mechanisms primarily disagree on the order of sulfoxidation and CdS bond formation. The first computa- tional study on the sulfoxide synthases used DFT to find that formation of the four-membered ring structure along Pathway II is the most likely oxidant to induce oxidation of the histidine ligand via PCET, which would then lead to formation of the CdS bond. However, this mechanism was calculated prior to the first crystal structure of EgtB and utilized a very limited set of residues for the calculation, and most notably, did not include Tyr377, which is known to play an important role in catalysis.133 A more recent computational study of EgtB calculated several mechanistic options along both Pathway I and Pathway II, finding that Pathway II is more favorable.134 This study proposed the formation of an S ¼ 2FeIII(superoxo) intermediate that attacks the sulfur via the distal oxygen atom followed by OdO bond cleavage, resulting in the formation of an FeIV(O)(sulfenate) intermediate. It was proposed that deprotonation of the TMH imidazole NdH followed by coupling of the sulfenate resulted in formation of the final sulfoxide product. However, this study found that imidazole NdH deprotonation was the rate determining step, and that there would be a solvent KIE, which is not in line with the experimental solvent KIE near unity.130 This study also predicted the incorrect stereochemistry for the final sulfoxide product.135 An alternate mechanistic pathway was proposed on the basis of QM/MM studies on EgtB (Fig. 17).136 It was proposed that Pathway I is more likely, suggesting that the first step involves an S ¼ 2FeIII(superoxo) that deprotonates Tyr377 via a Grotthuss mechanism, followed by rapid electron transfer from the tyrosinate to generate an FeIII(OOH) intermediate. The next step involves attack of the coordinated thiolate on the TMH C2 position and simultaneous electron transfer to Fe and proton transfer and electron transfer from the hydroperoxo ligand to Tyr377 tyrosyl radical, resulting in the formation of an FeII(superoxo) coordinated to the de-aromatized-TMH-g-GC sulfide. It is then proposed that the FeII(superoxo) intermediate abstracts an H-atom from the TMH unit Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes 19

Fig. 17 Reaction energy landscape for EgtB calculated by QM/MM. Reprinted with permission from reference Faponle, A. S.; Seebeck, F. P.; de Visser, S. P. J. Am. Chem. Soc. 2017, 139, 9259–9270. Copyright 2017 American Chemical Society. to rearomatize the imidazole and produce an FeII(hydroperoxo) intermediate. It is then found that the FeII(hydroperoxo) oxygenates the sulfide to produce the final product with no barrier (Fig. 17). Alternate mechanisms involving initial FedOdOdS formation were tested and were found to be less energetically accessible. The authors thus propose that proton-coupled electron transfer from Tyr377 is responsible for steering EgtB away from CDO-like S-dioxygenation reactivity. No computational studies on OvoA have been carried out. As a result, it is unclear if OvoA follows a similar or entirely different mechanistic pathway from EgtB. However, the mechanism proposed by Liao and Siegbahn does not line up with the lack of a KIE upon deuteration of the His susbtrate.122 Experimental studies have shown that addition of the thioether formed between histidine 125 and Cys to OvoA results in no oxidation following addition of O2 or H2O2, which is unexpected if Pathway I is operative. It is also not clear if the other newly discovered EgtB variants could follow alternative mechanisms for sulfoxide synthase reactivity.124 The sulfoxide synthases are a mechanistically complicated class of enzymes. More experimental and computational data are undoubtedly needed to better understand their reactivity patterns.

5 Persulfide Dioxygenase 5.1 Introduction Persulfide dioxygenases (PDOs) are enzymes that convert persulfides to their corresponding thiol and sulfite (Scheme 10). These enzymes are known in bacteria, plants, and humans, and are proposed to play essential roles in sulfur metabolism. In mammals, the persulfide dioxygenase, ETHE1, is found in mitochondria and is responsible for the second step in sulfide catabolism. In the first step, a sulfide:quinone converts H2S into persulfides, and ultimately pulls from the cellular reservoir of glutathione to generate glutathione persulfide. The second step uses ETHE1 to produce sulfite, regenerating glutathione. The third step involves a sulfur enzyme which reacts persulfides with the formed sulfite to produce thiosulfate, the final product in mitochondrial sulfur oxidation pathway.137

Scheme 10 20 Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes

Much of the interest in ETHE1 stems from the observation that patients with the rare autosomal recessive disease, ethylmalonic encephalopathy, exhibit defects in the gene that encodes ETHE1.138,139 This disease leads to neurological, cardiovascular, and – gastrointestinal defects, and is often lethal.138 140 It has been proposed that dysfunction of ETHE1 is associated with the buildup of 138 H2S leading to the inhibition of cytochrome c oxidase. A number of studies have found that plants contain a homolog of human ETHE1 that is important for plant metabolism.141 Persulfide dioxygenases have also been found in a large number of bacteria, and – may be related to the mechanisms by which pathogens display drug resistence.142 146 Thus, the persulfide dioxygenases represent a large and important family of enzymes that are widespread in nature.

5.2 Structure and Function The earliest studies on persulfide dioxygenases were with bacterial forms of the enzyme that were shown to convert elemental sulfur to sulfite in the presence of O2. Very little was known about these enzymes except that many required glutathione (GSH) for proper function, and that O2 was incorporated into the final sulfite product. In particular, little was known about the nature of the S-atom source to produce sulfite.147 It was not until a 2003 study that it was shown that the S-atom source in these S-oxidizing bacteria was glutathione persulfide (GSSH), which was generated in situ from the non-enzymatic reaction between elemental sulfur and glutathione.144 Characterization of human ETHE1 revealed that it binds one equivalent of Fe and has lower or no activity with other persulfides or thiols, such as cysteine persulfide, glutathione, and thiosulfate compared to GSSH.148 Some activity with CoA persulfide has been observed, but much lower than that with GSSH. It has also been shown from studies with plant ETHE1 that the enzyme is selective towards GSSH, exhibiting no activity with H2S, Cys, dithiothreitol, or 3-mercaptoethanol. However, it was found that 3-mercaptopyruvate can serve to transfer H2S to glutathione. A number of studies have also confirmed that O2 consumption was correlated with ETHE1 activity.138,141 The first crystal structure of a persulfide dioxygenase was reported in 2006 from the plant species Arabidopsis thaliana (Fig. 18).149 The protein structure was found to be a member of the metallo b-lactamase superfamily of proteins, which are typically enzymes found in antibiotic-resistant bacteria. However, this structure exhibited high sequence alignment with human ETHE1. The structure revealed an octahedral iron center bound by two histidine and one aspartate residue, similar to other nonheme iron oxygenases. However, the exact function of this enzyme was unclear. Further mechanistic studies with Arabidopsis thaliana ETHE1 revealed that one equivalent of Fe is bound to the protein and that the two histidines remain bound to the Fe center in solution.150 The structure for human ETHE1 was reported in 2014, and displayed very similar structural features to the plant ETHE1. The protein exists as a dimer and features Fe bound by two histidines and one aspartate.151 Docking simulations suggested that GSSH should be able to fit into the active site. An unpublished structure of the bacterial protein CstB from Staphylococcus aureus was shown to exhibit similar structural motifs to those seen in ETHE1 (PDB: 3R2U). This protein was later found to be a multidomain protein with PDO activity tightly coupled to persulfide transferase reactivity using a rhodanese domain. Interestingly, this PDO was found to utilize coenzyme A persulfide and bacillithiol persulfide as substrates.143 − The nature of the binding of the glutathione persulfide substrate to PDOs is currently unknown, as no structure for GSS bound − to a PDO has been determined, likely due to the instability of GSS . Insight into the substrate-bound form of PDOs has been obtained indirectly. A glutathione-bound structure was obtained from PDO found in Pseudomonas putida.145 This structure revealed that glutathione binds monodentate via the thiolate donor, replacing one of the resting state water molecules to afford an

Fe(His)2(Asp)(SR)(H2O) coordination geometry (Fig. 19). It was hypothesized that a similar monodentate sulfur ligation may

Fig. 18 Crystal structure showing overall protein structure of ETHE1 from Arabidopsis thaliana (left) and active site (right) in absence of GSSH substrate (PDB: 4CHL). Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes 21

Fig. 19 Crystal structure of active site of ETHE1 in the presence of glutathione (PDB: 4YSL).

− occur for the native substrate, GSS . The monodentate coordination is consistent with structures of cysteine persulfide and 3-mercaptopropionic acid persulfide fortuitously crystallized in CDO, which exhibit similar monodentate coordination.100

5.3 Mechanism Some specific residues in ETHE1 that are mutated in patients with ethylmalonic encephalopathy have been identified.139,152 Understanding the mechanisms of action, especially with relation to these mutations, has been a major focus of studies involving PDOs. One study found that mutation of R163 in human ETHE1 results in a lowered redox potential of the Fe center without significantly altering the protein structure.153 Another study has found that that same mutation leads to significantly lowered catalytic activity in the presence of substrates.154 The implications and relevance of these results to the mechanism of S-oxygenation are unclear, however. Mutants involving substitution of residues T152 and D196 resulted in a significant decrease in the stability and Fe binding affinity of the protein,148 but detailed mechanistic studies involving these mutations were not reported. Mutations to L55, C161, and T136 have also been shown to exhibit significant drops in catalytic activity.154 Based on the known S-oxygenation reactivity and the structure of ETHE1, the earliest proposed mechanism was in analogy to – the proposed mechanisms of S-oxygenation by CDO.148 This mechanism invokes coordination of the GSS to Fe, followed by III binding of O2 to generate an Fe (superoxo) intermediate. The distal oxygen atom attacks the coordinated sulfur, and subsequent OdO bond cleavage gives an FeIV(O) species bound to an S-oxygenated persulfide. Oxygen atom transfer from - the ferryl species leads to formation of thiosulfate (RSSO2), which is expected to undergo hydrolysis to produce glutathione and sulfite (Scheme 11). A similar mechanism was tested in a computational QM/MM study,155 which found glutathione persulfide binds bidentate to the Fe center via S-amide ligation. Binding of glutathione was followed by a series of O2 activation steps that mirror the reactivity proposed computationally for CDO156 (see Section 3.3). However, this study utilized a relatively limited set of protein residues and assumed bidentate coordination of the substrate, which seems less likely than monodentate coordination based on other studies.38,100,145

Scheme 11 22 Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes

Scheme 12

One intriguing study used g-glutamyl-homocysteinyl-glycine (GSHcySH) as a substrate analogue of GSSH.154 They hypothe- sized that that GSHcySH was topologically similar enough to GSSH to enable proper binding of the substrate and O2 activation, but the lack of a cleavable SdS bond would allow for the dioxygenated product prior to hydrolysis to be observed. Indeed, addition of GSHcySH resulted in single turnover and formation of the sulfinate product. This species remains bound to the Fe and prevents catalysis, indicating that the hydrolysis step in native catalysis is essential for turnover (Scheme 12). Spectroscopic studies indicated − that GSHcyS binds to the ferric center of ETHE1, as seen by the visible absorption band at 600 nm. Interestingly, glutathione generates a similar feature when added to ferric ETHE1, suggesting that it also binds to Fe. However, addition of O2 to ETHE1 incubated with GSH results only in the formation of disulfide (GSSG). It is possible that ETHE1 is capable of performing a single S-oxygenation of GSH to generate a sulfenate, which then can easily form disulfide in the presence of excess thiol. Alternatively, uncoupled autoxidation pathways could lead to the same product. Distinguishing these pathways would be interesting and relevant to the mechanism of S-oxygenation by PDO. MCD studies on human ETHE1 revealed significant changes to the ferrous active site upon binding of the persulfide substrate.38 The MCD spectrum of the persulfide bound Fe center was consistent with the presence of two species, most likely a 5-coord and II II 6-coord species. It was thus proposed that there is a mixture of 5-coordindate Fe (ON2)(SSR)(H2O) and 6-coordintate Fe (ON2) 82 (SSR)(H2O)2 centers, similar to what has been proposed for CDO. In this study NO was used as a probe for the initial O2 binding step in PDOs. Addition of NO led to the formation of a new EPR signal consistent with an S ¼ 3/2 {FeNO}7 species. DFT studies were able to reproduce spectral features of this {FeNO}7 and the same DFT model was used to predict the most likely bonding mode of the persulfide donor, the electronic structure of the hypothetical Fe(superoxo) intermediate expected to form upon addition of O2 to the ferrous center, and a possible mechanism for S-oxygenation (Fig. 20). The proposed mechanism for S-oxygenation by ETHE1 mirrors that proposed for CDO38,101 and is similar to that depicted in − III Scheme 11. In the first step, O2 binds to the ferrous center ligated to GSS as a monodentate ligand to form an S ¼ 2Fe (superoxo) species. Of note, docking simulations indicated that only monodentate coordination of the persulfide could be accommodated in the active site. The next step of the reaction is electrophilic attack of the persulfide S via the distal oxygen, and is rate limiting. The resulting FedOdOdS intermediate could then undergo a barrierless OdO bond cleavage step to generate an FeIV(O) which could then undergo rearrangement and O-atom transfer to generate the dioxygenated product. Hydrolysis releases thiol and produce the sulfite product. The same computational method was applied to CDO and predicts a similar reaction mechanism.101,102 More work is still needed to experimentally validate or provide alternatives to this proposed mechanism.

6 Synthetic Model Complexes

Studies involving synthetic analogues of the active sites of metalloenzymes provide the opportunity to directly correlate spectro- scopic properties and reactivity with the local environment surrounding the metal center.157 The amenability of model complexes towards synthetic modification allows their reactivity to be rationally tuned as a function of steric, geometric, and electronic properties. The feasibility of proposed enzymatic mechanisms can be assessed by determining the reactivity patterns of biomimetic model complexes. If proposed bond-making or bond-breaking events at the metal can be mimicked in a model system, the models can provide satisfying support for hypothetical mechanistic steps that are challenging, or sometimes impossible to probe directly in the biological systems. Similarly, a complete failure to reproduce a proposed enzymatic step in a well-defined model system may reveal that one or more hypothetical mechanistic steps are ill-conceived, and provide strong motivation for revising the mechanistic Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes 23

Fig. 20 Calculated mechanism and energy landscape for ETHE1 by DFT. Reprinted with permission from reference Goudarzi, S.; Babicz, J. T.; Kabil, O.; Banerjee, R.; Solomon, E. I. J. Am. Chem. Soc. 2018, 140, 14887–14902. Copyright 2018 American Chemical Society.

pathway in question. Model studies may also lead to the discovery of new modes of action for biologically relevant metal ions, forming the underpinnings of new mechanistic hypotheses for the analogous metalloenzymatic transformations.

Significant strides have been made in the synthesis of nonheme 3d metal complexes that react with O2, reduced O2 analogues (e.g., superoxide, peroxide), and O-atom donors to give metal/oxygen species of interest. However, studies of nonheme iron complexes in the presence of sulfur donors remains relatively scarce (Tables 1 and 2). The synthesis of these biomimetic complexes has relied on creative ligand design, which has allowed for the variation of electronic properties, steric profiles, hydrogen bonding, and other first- and second coordination sphere characteristics. In some cases, ligands have been synthesized to remain structurally faithful to coordination modes seen for specific enzyme active sites. Other approaches rely on donor sets that deviate from those strictly observed in nature, but still incorporate donors that impart relevant biomimetic properties. Designs include either polydentate (Scheme 13) or mixed ligand sets (Scheme 14).

6.1 Sulfur Oxygenation Early work on modeling CDO employed a bis(imino)pyridine-derived framework with a pendant thiolate donor (Scheme 15). II Structural determination of the complex Fe (LN3S)(OTf ) (1) by single crystal X-ray diffraction (XRD) shows that it is five- coordinate, and consists of three neutral N donors and a thiolate donor cis to the potential O2 on the iron center. II II + The reaction of Fe (LN3S)(OTf ) with O2 resulted in the formation of the triply oxygenated sulfonate species, [Fe (LN3SO3)] , which was characterized by electrospray ionization mass spectrometry (ESI-MS, 532.1 m/z). Isotopic labeling studies that involved a 18 18 combination of O2 and H2 O confirmed that the sulfonate O atoms originated from O2 gas. A mixed isotope experiment 18 16 ( O2/ O2) confirmed that triple oxygenation at sulfur occurs via a sequential dioxygenation-monooxygenation mechanism. II Reaction of O2 with the non-redox-active Zn analogue, Zn (LN3S)(OTf ), results in no S-oxygenation, supporting a mechanism for Fe that involves binding and activation of O2 by the redox-active Fe center. Further support for this hypothesis came from computational studies, which suggested that a mechanism similar to that proposed for CDO (Scheme 8, Mechanism I) is feasible. II However, for Fe (LN3S)(OTf ), the proposed rate-determining step is OdO bond cleavage rather than SdO bond formation, which 156 III •− was predicted to be rate-determining in the enzyme. The characterization of iron-dioxygen adducts, such as an Fe (O2 ) species, was unsuccessful. Another independent computational study compared these model complexes with a hypothetical selenium- substituted analogue.179 They compared the hypothetical four membered FedOdOdSR and FedOdOdSeR ring structures and 24 Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes

Table 1 Structural and spectroscopic properties of selected nonheme, sulfur-ligated ferrous complexes.

III II Coord. sphere d(FedS) Spin state d (│DEQ│)E1/2 (Fe /Fe ) Reference (Å) mm s–1 V vs. Fc+/Fc

II a Fe (LN3S)(OTf ) (1)N3SO 2.2942(8) S ¼ 2 n.r. n.r. [158] II [Fe (LN3S)(py)]OTf (2)N4S 2.3246(18) S ¼ 2 n.r. n.r. [159] II b [Fe (LN3S)(DMAP)]OTf (3)N4S 2.3246(18) S ¼ 2 0.87 (2.70) n.r. [159] II 0 b [Fe (LN3S)(DMAP)] (4)N4S 2.2179(5) S ¼ 1/2 0.33 (2.04) n.r. [159] iPr II b c ( BIP)Fe (SPh)(Cl) (7)N3SCl 2.3305(9) S ¼ 2 0.78 (2.62) –0.173 [159,160] iPr II c ( BIP)Fe (SPh)(OTf ) (8)N3SO 2.2795(9) S ¼ 2 n.r. –0.372 [160] II d e [Fe (N3PyS)(CH3CN)]BF4 (9a)N5S 2.3018(4) S ¼ 0 0.14 (0.26) –0.226 [161] II d f [Fe (N3PyS)(MeOH)]BF4 (9b)N4SO n.r. S ¼ 2 1.05 (2.77) –0.583 [161,162] II e Fe (N3PySO2))(SCN) (10)N5SO 2.1812(9) S ¼0 n.r. –0.001 [161] Me,Ph [Tp FeCysOEt] (12)N4S 2.3122(9) S ¼ 2 n.r. n.r. [163] Me,Ph Tp FeCysAm (13)N4S 2.3175(6) S ¼ 2 n.r. n.r. [164] Ph2 [Fe( TIP)(CysOMe)]BPh4 (14)N4S 2.3107(6) S ¼ 2 n.r. n.r. [165] Ph2 [Fe( TIP)(CysAm)]BPh4 (15)N4S 2.3051(5) S ¼ 2 n.r. n.r. [165] II d Fe (Me3TACN)(abt)(OTf ) (16)N4SO 2.4352(4) S ¼ 2 1.07 (3.55) n.r. [82] II CF3 d Fe (Me3TACN)(abt )(OTf ) (17)N4SO 2.4343(4) S ¼ 2 1.08 (3.20) n.r. [82] II d [Fe (iPr3TACN)(abt)](OTf ) (18)N4S 2.3573(4) S ¼ 2 0.93 (1.97) n.r. [82] II CF3 d [Fe (iPr3TACN)(abt )](OTf ) (19)N4S 2.3753(6) S ¼ 2 0.95 (2.06) n.r. [82] II Me2 e [Fe (S N4(tren))](PF6)(20)N4S 2.328(1) S ¼ 2 n.r. –0.482 [166] II Me2 e,f [Fe (S N4(tren-Et4))](PF6)(22)N4S 2.317(1) S ¼ 2 n.r. 0.028 [166] II II Ni LFe (CH3CN)(Z-C5Me5)]BPh4 (23)C3S2N 2.2850(6) S ¼ 0 n.r. n.r. [167] 2.2863(6) II II d e Ni LFe (EtCN)(Z-C5Me5)]BPh4 (24)C3S2N 2.2867(6) S ¼ 0 0.55 (2.10) −0.57 167 2.2839(6) Me2 [Fe(Tp )(2-ATP)] (26)N4S 2.3107(4) S ¼ 2 n.r. n.r. [168] II Me2 [Fe (S2 N3(Pr,Pr))] (28)N3S2 2.3306(5) S ¼ 1 n.r. n.r. [169] 2.3263(5) II g h e Fe (Me3TACN)(S2SiMe2)(33)N3S2 2.371(2) S ¼ 2 0.93 (2.30) , –0.6 [170,171] 2.410(2) II amide g [Fe (N3Py SR)](BF4)2 (37)N4S(thioether)O 2.2911(6) S ¼ 0 0.44 (0.77) n.r. [172] II amide 2+ [Fe (N3Py S(O)R)] (BF4)2 (39)N4S(thioether)O 2.254(13) n.r. n.r. n.r. [172] II g e [Fe (TMCS)](PF6)(40)N4S 2.297(3) S ¼ 2 0.90 (3.0) 0.148 [173,174] II + [Fe (cyclam-PrS)] (47)N4S 2.286(1) S ¼ 2 n.r. n.r. [175] II c,f [Fe ([15]aneN4)(SC6H5)]BF4 (50a)N4S 2.3316(11) S ¼ 2 n.r. 0.076 [176,177] II c,f [Fe ([15]aneN4)(SC6H4Cl)]BF4 (50b)N4S 2.3197(12) S ¼ 2 n.r. 0.156 [176] II c,f [Fe ([15]aneN4)(SC6H4OMe)]BF4 (50c)N4S 2.3240(17) S ¼ 2 n.r. 0.021 [176] II c,f [Fe ([15]aneN4)(SC6F4SC6F5)]BF4 (50d)N4S 2.3426(8) S ¼ 2 n.r. 0.388 [176] II c,f [Fe ([15]aneN4)(SC6F4NO2)]BF4 (50e)N4S 2.3305(15) S ¼ 2 n.r. 0.223 [176] II h [Fe (N3PySR)(CH3CN)](BF4)2 (52)N5S (thioether) 2.2848(4) S ¼ 0 0.44 (0.55) n.r. [162,178] an.r. indicates not reported. bData collected as a solid at 5 K in the presence of a 47 mT magnetic field. c CV data collected in CH2Cl2. dData collected with zero field as a solid at 5 K. e CV data collected in CH3CN. fAnodic peak potential reported. g Data collected in CH3CN at 5 K in the presence of a 47 mT magnetic field. hData collected with zero field at 80 K.

found that the -SR donor was better at donating electron density necessary to cleave the OdO bond than -SeR. The authors hypothesized that this ability to cleave the OdO bond is why selenocysteine is not oxygenated by CDO. II Addition of either pyridine or 4-dimethylaminopyridine (DMAP) to Fe (LN3S)(OTf ) generates a new high-spin ferrous II II 1− 180 complex, [Fe (LN3S)(py)]OTf (2) or [Fe (LN3S)(DMAP)]OTf (3), each with an [N4S] coordination environment. Reduction II 0 of 3 with Na/Hg amalgam produced a new S ¼ 1/2 species [Fe (LN3S)(DMAP)] (4), best described as an S ¼ 1 Fe center antiferromagnetically coupled with a ligand-based radical. The reactivity of 2, 3, and 4 were compared, and it was found that 2 II + and 3 performed S-oxygenation, producing [Fe (LN3SO3)] , as determined by mass spectrometry. However, 4 performed both Fe and S oxygenation, producing a mixture of S-oxygenates and m-oxo dimer products, as seen by mass spectrometry. Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes 25

Table 2 Structural and spectroscopic properties of selected nonheme sulfur-ligated Fe/oxygen complexes.

Complex Coordination Spin lmax, (e) n(OdO) n(FedO) d (│DEQ│) EPR Sphere State nm (M–1 cm–1)cm–1 cm–1 mm s–1

Superoxo Me2 a b [Fe(O2)(Tp )(2-ATP)] (27) N4SO S 1 490 (1200), 655 (1800), 1135, 504 n.r. Silent 860 (2200) 1105 III Me2 c [Fe (S2 N3(Pr,Pr))(O2)] (29) N3S2O S ¼ 0 409, 520, 707 1122, n.r. n.r. Silent 1093 Peroxo II IV 2 5 [Ni LFe (Z -O2)(Z -C5Me5)]BPh4 C3S2O2 S ¼ 0 410 940 n.r. 0.42 (0.33) n.r. (25)d III - e [Fe (O2)(TMCS)] (49) N4SO S ¼ 5/2 460 (6100), 610 (1200) n.r. n.r. 0.71 (-1.9) 7.9, 5.75 f Fe2(O2)(Me3TACN)2(S2SiMe2)2 (34) N3S2O n.r. 300 (9000), 390 (5000) 849 n.r. 0.53 (0.77) Silent 530 (2050), 723 (3600) Hydroperoxo III g [Fe (cyclam-PrS)(OOH)]PF6 (48) N4SO S ¼ 5/2 530 (1400) 891 419 n.r. 7.72, 5.40, 4.15 III amide [Fe (OOH)(N3Py SR)](BF4)2 N4S(thioether) S ¼ 1/2 567 (900) 800 612 n.r. 2.17, 2.16, (54a)h O 1.95 III h [Fe (OOH)(N3PySR)](BF4)2 (53a) N4S(thioether) S ¼ 1/2 542 (1000) 809, 787 615 – n.r. 2.17, 2.11, O 664 1.97 III Me2 c [Fe (S2 N3(Pr,Pr))(OOH)] (31) N3S2O S ¼ 1/2 696 n.r. n.r. n.r. 2.23, 2.15, 2.00 III Me2 + i [Fe S N4(tren)(OOH)] (45) N4SO S ¼ 1/2 452 (2780) 788, 781 n.r. n.r. 2.14, 1.97 Alkylperoxo III t [Fe ([15]aneN4)(SC6F4NO2)(OO Bu)] N4SO S ¼ 1/2 522 (3100) 800 615 n.r. n.r. j BF4 (51e) III [Fe ([15]aneN4)(SC6F4SC6F5) N4SO S ¼ 1/2 508 (3000) 799 623 n.r. n.r. t j (OO Bu)]BF4 (51d) III t [Fe ([15]aneN4)(SC6H4Cl)(OO Bu)]BF4 N4SO S ¼ 1/2 524 (2100) 803 612 n.r. 2.19, 1.97 (51b)j III t [Fe ([15]aneN4)(SC6H4OMe)(OO Bu)] N4SO S ¼ 1/2 514 (1900) 802 608 n.r. n.r. j BF4 (51c) III t [Fe ([15]aneN4)(SC6H5)(OO Bu)]BF4 N4SO S ¼ 1/2 526 (2150) 803 612 n.r. 2.20, 1.97 (51a)j III t amide [Fe (OO Bu)(N3Py SR)](BF4)2 N4SO S ¼ 1/2 620 (2000) 796 700 n.r. 2.17, 2.11, (54b)k 1.96 III t k [Fe (OO Bu)(N3PySR)](BF4)2 (53b) N4SO S ¼ 1/2 600 (1670) 796 691 n.r. 2.14, 2.08, 1.96 Oxo IV amide l [Fe (O)(N3Py SR)](BF4)2 (38) N4S(thioether) S ¼ 1 750 (400) – n.r. 0.04 (0.80) Silent O IV f Fe (O)(Me3TACN)(S2SiMe2)(35) N3S2O S ¼ 1 385 (2600), 460 (1500), – 735 0.21 (1.54) Silent 890 (390) IV m [Fe (O)(TMCS)](PF6)(41) N4SO S ¼ 1 460 (1300), 570 (1100), – n.r. 0.19 (- Silent 850 (230) 0.22) aFrom reference [168]. bn.r. indicates not reported. cFrom reference [169]. dFrom reference [167]. eFrom reference [192]. fFrom reference [171]. gFrom reference [175]. hFrom reference [178]. iFrom reference [166]. jFrom references [176, 177]. kFrom reference 172. lFrom reference [172]. mFrom reference [174]. 26 Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes

Scheme 13

II II Structurally related thioether complexes [Fe (LN3SMe)(H2O)3](OTf )2 (5), and [Fe (LN3SMe)Cl2](6) were also prepared and 181 were shown to perform S-oxygenation reactivity. In this case, high temperatures (up to 60 C) were required to initiate O2 2+ reactivity. Oxygenation of 5 resulted in the formation of the sulfoxide product, [Fe(LN3S(O)Me)(OTf )] and oxygenation of 6 2+ 1 generated the sulfone, [Fe(LN3S(O2)Me)Cl] , as determined by mass spectrometry and by H NMR spectroscopic analysis of the hydrolyzed organic products. The mechanism of S-oxygenation was not discussed, but these results suggest that the nature of the supporting ligands can control the extent of S-oxygenation (i.e., mono- versus di-oxygenation). The related complexes (iPrBIP)FeII(SPh)(Cl) (7) and (iPrBIP)FeII(SPh)(OTf ) (8) featured a similar bis(imino)pyridine frame- work but with an untethered arylthiolate donor. These complexes were structurally characterized and both contain 5-coordinate hs-FeII centers; however, a striking difference between these related complexes is the orientation of the thiolate donor with respect to iPr II the potential open coordination site for binding of O2.In( BIP)Fe (SPh)(Cl), the phenylthiolate ligand is trans to the possible O2 binding site, whereas in (iPrBIP)FeII(SPh)(OTf ), the phenylthiolate ligand is cis. For the trans-thiolate ligated complex, addition of

O2 leads to the formation of disulfide. Laser desorption ionization mass spectrometry (LDIMS) analysis of the reaction mixture IV iPr + 18 revealed a dominant peak consistent with the Fe (O) formulation, [( BIP)Fe(O)(Cl)] , which shifted by +2 units upon O2 IV substitution. Reaction of this species with PPh3 gave OPPh3 in good yield, consistent with the oxidized Fe (O) formulation. In contrast, addition of O2 to the OTf complex with a thiolate donor cis to the open binding site leads to the formation of benzenesulfonic acid, based on organic product analysis by 1H NMR spectroscopy and reversed phase high performance liquid Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes 27

Scheme 14

M=Zn M=Fe O O TfO S TfO S O2 O2 O M Ar Fe No Reaction 18 CH2Cl2 N N H2 O N N 23 °C N CH2Cl2 N 23 °C

1: M =Fe Scheme 15

Scheme 16 chromatography (HPLC). These studies suggest that the orientation of the thiolate donor in the FeII starting material plays an essential role in determining the ultimate selectivity of Fe versus S-oxygenation in these model complexes (Scheme 16). A computational study182 found that OTf must bind trans to the open coordination site in 8 due to an unfavorable steric interaction with the BIP ligand framework, whereas Cl is able to bind either cis or trans. Optimizing the four putative and hypothetical iPr III Fe(superoxo) structures ( BIP)Fe (SPh)(X)(O2) (where X ¼ Cl and OTf ligated cis or trans) indicated that the thiolate ligand dissociates as a thiyl radical when bound trans to the iron center. These calculations support the observed experimental requirement for cis ligation of the thiolate donor for S-oxygenation reactivity. 28 Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes

Scheme 17

II Following the O2 chemistry of the BIP complexes was significantly hampered by the intense absorption bands for BIP-Fe species −1 −1 iPr II (e.g., lmax ¼ 715 nm (e 4000 M cm ) for ( BIP)Fe (SPh)(OTf )), making it very difficult to identify any putative O2 1− intermediates. Several of these BIP-derived model complexes also did not incorporate the complete [N4S] coordination environ- ment present in substrate-bound CDO. Such a coordination environment is presented to the metal by the N3PyS ligand shown in – II Scheme 17. Reaction of N3PyS and [Fe (H2O)6](BF4)2, or metalation of the sulfur-protected N3PySEtCN followed by deprotec- 183 II tion with added base results in the formation of [Fe (N3PyS)(CH3CN)]BF4 (9a)(Scheme 17), which was characterized by XRD. II The crystal structure of [Fe (N3PyS)(CH3CN)]BF4 reveals a six-coordinate Fe center with a potentially labile solvent ligand bound cis II to the thiolate donor. This Fe complex is low-spin (S ¼ 0) in CH3CN, and does not react with O2 in this solvent. However, upon dissolution in methanol, the FeII complex undergoes a color change indicating conversion to a high-spin (S ¼ 2) species. Addition II of O2 in CH3OH leads to the consumption of the Fe complex and formation of a new green species. Addition of KSCN to this green II II species leads to single crystals of Fe (N3PySO2)(SCN) (10) (XRD), a doubly oxygenated, Fe (sulfinate). The reaction of high-spin II + [Fe (N3PyS)(CH3OH)] (9b) with O2 provides a rare functional model of the thiol dioxygenase enzymes, in which the sulfur center in FeII(SR) is preferentially oxygenated over the iron center. Unlike the BIP complexes, the N3PyS ligand allows for the observation of a new green species with lmax ¼ 674 nm upon oxygenation. However, this species could not be identified. II Nitric oxide was employed as an O2 surrogate with [Fe (N3PyS)(CH3CN)]BF4. Addition of NO results in the formation of a new {FeNO}7 species (11), which was characterized by EPR, Mössbauer, XAS, UV-vis, and NMR spectroscopies.184 Complex 11 exhibits a low-spin (S ¼ ½) ground state, the same as that seen for the NO adduct of CDO, but which contrasts the S ¼ 3/2 ground state typically observed for other nonheme {FeNO}7 enzymes.95,185,186 This {FeNO}7 species releases NO upon illumination with white III II + light, and under aerobic conditions, leads to nitric oxide oxidation to give an Fe (nitrite) complex. In addition, [Fe (N3PyS)(NO)] could be interconverted between {FeNO}6 and {FeNO}8 species by one electron, outer-sphere oxidation and reduction, respec- – tively (Scheme 18).183,187 These complexes represent a rare series of {FeNO}6 8 complexes stabilized within the same ligand environment, and provide a unique example of such a series with thiolate ligation.185,186,188 The oxidized {FeNO}6 complex readily undergoes photolytic, thermal, or solvent-mediated NO dissociation.187 The reduced {FeNO}8 complex provides a rare example of 183 a nitric oxide adduct that spontaneously produces N2O upon thermal decay, which has implications for possible mechanisms of the nitric oxide reductases (NORs). These reactivity patterns suggest the inclusion of the unique thiolate donor has an influence on promoting unusual nonheme iron transformations with nitric oxide. Me,Ph A complex utilizing the facially-coordinating N3 ligand, hydrotris(pyrazolyl)borate, and cysteine ester, Tp FeCysOEt (12), 163 was prepared as a model of CDO. Addition of O2 to 12 in CH2Cl2 resulted in slow decomposition and the formation of the

Scheme 18 Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes 29

Fig. 21 S-oxygenation reactivity by Tp and TIP cysteine ester and cysteamine complexes and time-resolved UV-vis spectra for reaction of 14 with O2. Plot adapted with permission from reference Fischer, A. A.; Stracey, N.; Lindeman, S. V.; Brunold, T. C.; Fiedler, A. T. Inorg. Chem. 2016, 55, 11839–11853. Copyright 2016 American Chemical Society. sulfinate product following workup and analysis by 1H NMR spectroscopy (Fig. 21). Mass spectrometry studies with mixtures of 16 18 O2/ O2 clearly indicated that both O-atoms from O2 were incorporated into the sulfinate product. Though the Fe(sulfinate) product could not be definitively characterized by XRD, XAS data and DFT calculations were consistent with a model involving formation of an Z2-O,O bound Fe(sulfinate) product. Further studies showed that the analogous cysteamine complex, TpMe, PhFeCysAm (13), could be prepared as a model of ADO. As with cysteine ethyl ester, dioxygenation of the cysteamine to taurine was observed upon workup of the reaction of 13 with excess O2. However, no Fe/oxygen intermediates or Fe-containing products were reported. Related iron(II) complexes containing the neutral N3 ligand tris(imidazolyl)phosphine (TIP) and cysteine methyl ester were Ph2 Ph2 165 prepared, with formulas [Fe( TIP)(CysOMe)]BPh4 (14) and [Fe( TIP)(CysAm)]BPh4 (15)(Fig. 21). These complexes contain high spin ferrous centers with the expected bidentate coordination of the S/N ligands. As was seen for the former Tp −4 −1 II complexes, addition of O2 results in slow decay (k1(20 C) ¼ 1.9(4) 10 s for 14), of the Fe starting material and formation of sulfinate products. Kinetics studies of the oxygenation reaction indicated that the N3 ligand has only minor effects on the rate of the reaction. Calculations on the reaction trajectories for both the Tp and TIP complexes suggested that similar mechanisms were operative, with a rate-determining step involving SdO bond formation via an FeIII(superoxo) intermediate. A larger degree of thiyl radical character for the TIP versus the Tp complexes was calculated by DFT, leading to the hypothesis that the neutral N donors in the enzyme (3 His) may be important in promoting thiyl character and a lower barrier for SdO bond formation. As with the Tp complexes, no Fe/O2 intermediates were experimentally observed. Thus NO was added as an O2 surrogate. Formation of high-spin {FeNO}7 (S ¼ 3/2) species was observed, in contrast to the low-spin (S¼1/2) ground state FeNO adducts seen in CDO. However, the binding of NO suggests that O2 may bind directly to the Fe center in the model complexes prior to S-oxygenation. II II The iron(II) 1,4,7-triazacyclononane (TACN) derivatives [Fe (Me3TACN)(CH3CN)3](OTf )2 and Fe (iPr3TACN)(OTf )2 were II 82 employed to synthesize the [Fe (N4S(thiolate))] complexes shown in Fig. 22. These complexes have the advantage of labile sites

Fig. 22 (A) Syntheses of complexes 16–19. (B) Zero-field 57Fe Mössbauer spectra of 16–19 collected at 5 K. Plot adapted with permission from reference Gordon, J. B.; McGale, J. P.; Prendergast, J. R.; Shirani-Sarmazeh, Z.; Siegler, M. A.; Jameson, G. N. L.; Goldberg, D. P. J. Am. Chem. Soc. 2018, 140, 14807–14822. Copyright 2018 American Chemical Society. 30 Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes

CF3 on the metal for chelation of bidentate NS donors similar to cysteine. Addition of [Et3NH][abt] or [Et3NH][abt ] (abt ¼ II II 2-aminobenzenethiolate) to the Fe (TACN) precursors led to the isolation and structural characterization of Fe (Me3TACN)(abt) II CF3 II II CF3 (OTf ) (16), Fe (Me3TACN)(abt )(OTf ) (17), [Fe (iPr3TACN)(abt)](OTf ) (18), and [Fe (iPr3TACN)(abt )](OTf ) (19)in II good yields. The two Me3TACN complexes are six-coordinate (6-coord) Fe (N4SO) centers, with a triflate anion occupying the II sixth site, and the iPr3TACN complexes are five-coordinate (5-coord) Fe (N4S) centers with the triflate counterion sitting outside the coordination sphere. Analysis by 57Fe Mössbauer spectroscopy on solid samples of complexes 16–19 reveals an interesting trend for –1 these high-spin ferrous centers. The 5-coordinate complexes 18–19 exhibit a quadrupole splitting of |DEQ | 2mms , whereas the –1 6-coordinate complexes 16–17 exhibit a much larger |DEQ | > 3mms . Trends between coordination number and the quadrupole splitting of structurally well-defined Fe complexes are difficult to establish. The complexes in Fig. 22 show that 5- versus

6-coordinate iron(II) complexes can be readily distinguished by their |DEQ | values in a series of structurally related complexes. The spectroscopic trends exhibited by the series of complexes in Fig. 22 were useful in helping to assign the different species seen in the Mössbauer spectrum of the substrate-bound CDO enzyme. There are two closely overlapping doublets observed in the II –1 –1 Mössbauer spectrum of Cys-bound Fe CDO, with parameters modeled as d ¼ 1.03 mm s ,|DEQ | ¼ 2.28 mm s (species A) and d –1 –1 0 0 ¼ 1.10 mm s ,|DEQ | ¼ 3.14 mm s (species A )(Section 3.2). Early computational modeling (DFT) predicted that A was 6-coord II II with H2O in the open site (Fe (His)3(Cys)(H2O)), while A was the 5-coord center, Fe (His)3(Cys). However, comparison with the trends established for the model complexes combined with further DFT calculations indicated that the two quadrupole doublets observed for Cys-bound CDO likely correspond to water bound and unbound forms of the Fe center, with A assigned as the 5-coordinate, water-bound complex and A0 as the 6-coordinate Fe center. Reaction of 16 with excess O2 at −95 C results in a transient blue species that converts to a red species upon warmup to 23 C. Analysis by 1H, 13C, and 1H–13C HSQC NMR spectroscopies and electron ionization mass spectrometry (EI-MS) revealed the formation of the S-oxygenated product methyl 2-aminobenzenesulfinate (42%). An initial sulfinate species likely forms, followed 18 by esterification in MeOH to give the final sulfinate ester. Employing O2 showed that one oxygen of the sulfinate derivative originates from O2. Adding O2 to the CF3-substituted 17 at −95 C also results in S-oxygenation. In this case, however, the Fe-bound sulfinate product is isolated as a (m-oxo)diferric complex with bridging sulfinate ligands, characterized by XRD (Scheme 19). Mass spectrometry studies confirmed that the four sulfinate O-atoms in the diferric product were derived from O2. Addition of the non-native abt substrate followed by O2 to a mutant of the enzyme, C93G CDO, led to disulfide as the major product. Very small changes were observed in the Mössbauer spectra of C93G CDO upon addition of the abt substrate, suggesting only weak (or no) binding to the Fe in the enzyme. Thus, unlike in the model complexes, proper chelation of abt does not occur in the enzyme, preventing S-oxygenation. This combined enzyme/model study with abt suggests that N/S substrates need to be chelated properly to the Fe center in CDO to undergo S-oxygenation. This requirement likely contributes to the substrate specificity observed for CDO (see Section 3.3).

6.2 Fe/O2 Intermediates

II Me2 The 5-coordinate, N4S-ligated ferrous complex [Fe (S N4(tren))](PF6)(20) reacts with O2 to produce a m-oxo dimer, III Me2 189 [Fe (S N4(tren))]2(m-O)(PF6)2•3MeCN (21)(Scheme 20), which was characterized by X-ray crystallography. The reaction required 0.25 equiv of O2, a stoichiometry that is consistent with mechanisms observed for the formation of Fe(porphyrin) m-oxo III III IV 190 dimer complexes involving initial Fe (superoxo), Fe2 (peroxo) and Fe (oxo) intermediates. An intermediate with an absorp- tion feature at 460 nm was observed in MeOH when O2 is added to 20 at −78 C. The authors did not elaborate further on the identity of this intermediate. The electronic properties and reactivity of the (m-oxo) dimer complex 21 were examined. It was found − that the ferric centers were weakly anti-ferromagnetically coupled with J ¼ −28 cm 1. Addition of acids led to cleavage of the oxo- bridged dimer, which could be reformed upon addition of a hydroxide source. To prevent the formation of an oxo-bridged dimer, a II Me2 more sterically encumbered complex, [Fe (S N4(tren-Et4))](PF6)(22) was prepared. However, 22 did not react with O2 (Scheme 20). Cyclic voltammetry of 20 and 22 showed that 20 exhibits a reversible redox couple (E1/2 ¼ −100 mV versus SCE), although 22 exhibited only an irreversible wave at a much higher potential (+410 mV versus SCE). It was hypothesized that the FeIII center could

Scheme 19 Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes 31

Scheme 20

II II Fig. 23 Catalytic reduction of O2 to H2ObyNiLFe (RCN)(Z-C5Me5)]BPh4. not be accessed in the latter complex because it would produce shorter MdL bond lengths which would create unfavorable steric interactions with the bulky ethyl groups. IV II II A dithiolate-ligated Fe/O2 adduct, assigned as an Fe (peroxo), was prepared by addition of O2 to Ni LFe (RCN)(Z-C5Me5)] 0 191 BPh4 (23–24)(L¼N,N -diethyl-3,7-diazanonane-1,9-dithiolato, R¼CH3 (23)orEt(24)) at –40 C in acetonitrile (Fig. 23). The II IV 2 5 product [Ni LFe (Z -O2)(Z -C5Me5)]BPh4 (25) was crystallographically characterized, and the OdO bond distance (1.381(3) Å), –1 18 16 –1 as well as the n(OdO) of 940 cm (Dn( O2– O2) ¼ –53 cm ) as seen by IR spectroscopy, are in the range for metal peroxo 18 species. Furthermore, addition of H2 O2 resulted in exchange of the peroxo core, consistent with the peroxo assignment. Magnetic susceptibility measurements on 25 indicated an S ¼ 0 ground state. Analysis of 25 by 57Fe Mössbauer spectroscopy revealed a –1 –1 doublet with d ¼ 0.42 mm s and |DEQ | ¼ 0.33 mm s . It was found that 23 and 24 were capable of reducing O2 to water upon addition of a reductant and proton source, regenerating ferrous starting material. The ferrous complex, [Fe(TpMe2)(2-ATP)] (26) was prepared as a structural model of the active site of CDO, using the non-native 168 substrate 2-aminobenzenethiolate (abt or 2-ATP). Addition of O2 in THF at −80 C resulted in the formation of a new purple, metastable, EPR-silent species (27) with UV-vis features at 490, 655, and 860 nm (Scheme 21). Interestingly, the presence of the two 81 35 bands with lmax > 500 nm resembles those seen for Fe/O2 intermediates trapped in CDO and IPNS. This intermediate had a half-life of 10 min, and gradually decayed to a new EPR active species. The resonance Raman spectrum of this intermediate revealed −1 18 −1 −1 18 16 −1 O2-sensitive peaks at 1105 and 1135 cm ( O2, 1055 cm ). An additional feature at 504 cm (Dn( O2– O2) ¼ –16 cm ) was − also observed. The bands at 1105 and 1135 cm 1 are in the typical range seen for the n(OdO) of metal-superoxo species, and the − feature at 504 cm 1 was assigned as n(FedO), leading to the assignment of 27 as a mononuclear FeIII(superoxo) species. Time- dependent DFT (TD-DFT) calculations supported the assignment. A caveat is that the Mössbauer spectrum for 27 was not reported, which would help determine the purity and yield of the superoxo adduct. Decay of 27 resulted in the formation of an EPR active 32 Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes

Scheme 21

PF O 6 H H O H N N N O2 N N KO2 N FeII FeIII FeIII N S THF N S THF N S S −73 °C S −73 °C S

28 29 30 O −3 −1 H kobs= 1.7 x 10 min THF O2 − 8 25 °C O 7.65 d -THF

2 + −7.75 R =0.981 OH THF

H H O ) O 0 N N N N ] 2 −7.85 2 FeIII FeIII R =0.987 100 eq CHD −3 −1 N S N S kobs= 8.2 x 10 min S S −7.95 ]/[Fe-O 2 −2 −1 kobs= 1.56 x 10 min 32 31 −8.05

In([Fe-O −8.15 R2=0.995

−8.25 0 5 10 15 20 25 30 35 Time (min)

III Me2 Fig. 24 Formation and reactivity of [Fe (S2 N3(Pr,Pr))(O2)]. Plot reprinted with permission from reference Blakely, M. N.; Dedushko, M. A.; Yan Poon, P. C.; Villar- Acevedo, G.; Kovacs, J. A. J. Am. Chem. Soc. 2019, 141, 1867–1870. Copyright 2019 American Chemical Society. species which was proposed to be an FeIII(anilinide) complex. No S-oxygenation of the substrate was observed, and the disulfide was the only detectable organic product followed by warm-up to room temperature. II II Me2 The dithiolate-ligated Fe complex, [Fe (S2 N3(Pr,Pr))] (28) reacts with dioxygen in THF at −73 C to give a new metastable 193 species 29 with UV-vis bands at 409, 520, and 707 nm (Fig. 24). Resonance Raman studies revealed O2-sensitive peaks at 1093 –1 III Me2 + and 1122 cm consistent with superoxo n(OdO) modes, and addition of KO2 to the ferric complex [Fe (S2 N3(Pr,Pr))] (30) III III Me2 also results in the formation of 29. Complex 29 was thus assigned as an Fe (superoxo) species, [Fe (S2 N3(Pr,Pr))(O2)]. The decay of 29 at –73 C gives a new species 31 (87%, EPR quantitation) with a rhombic EPR spectrum (g ¼ [2.23, 2.15, 2.00]). When

THF-d8 is substituted for THF, the rate of decay is slower, giving a KIE of 4.8, and implicating an H-atom abstraction step in the decay of 29–31. A significant increase in the rate of decay is observed upon addition of the weaker CdH bond substrate 1,4- cyclohexadiene. These observations led the authors to propose that 31 is an Fe(OOH) complex, generated from THF oxidation –1 (BDE ¼ 92 kcal mol ). At higher temperature (25 C), addition of O2 to 28 results in the formation of the previously synthesized III 2 Me2 Me2 + sulfenate complex, [Fe (Z -S O)(S N3(Pr,Pr))] (32), in which the sulfenate O atom was derived from O2. Together these results suggest that an Fe(OOH) is formed en route to the S-oxygenation of the thiolate ligand. II 194 171 The dithiolato complex Fe (Me3TACN)(S2SiMe2) (33) immediately reacts with O2 at 23 C to give intractable products. However, an Fe/O2 intermediate can be stabilized by significantly lowering the reaction temperature to −135 C in 2-MeTHF. Intense UV-vis features at 300, 390, 530, and 723 nm appear, and EPR spectroscopy showed that this green species is EPR silent. –1 –1 Mössbauer spectroscopy revealed a single, sharp quadrupole doublet with d ¼ 0.53 mm s and │DEQ│ ¼ 0.77 mm s , and – resonance Raman spectroscopy showed a n(OdO) at 849 cm-1 (D18O ¼ –49 cm 1). These data pointed to the assignment of this low temperature O2 adduct as a peroxodiiron species, Fe2(O2)(Me3TACN)2(S2SiMe2)2. Characterization by XAS provided EXAFS data that corroborated this structure, giving an FedFe distance between 4.4–4.7 Å. This species is highly photosensitive as well as Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes 33

Scheme 22 thermally sensitive, and exposure to light or warming of 34 above –120 C leads to the isosbestic decay of 34 and formation of a second intermediate with UV-vis bands at 385, 460 and 890 (e390) nm. The relatively weak absorbance at 890 nm is characteristic of an FeIV(O) (S ¼ 1) species, suggesting that 34 undergoes OdO bond homolysis to produce an FeIV(O) complex, FeIV(O) –1 (Me3TACN)(S2SiMe2)(35). Mössbauer spectroscopy of 35 revealed a quadrupole doublet with d¼0.21 mm s and│DEQ│ ¼ 1.54 – mm s 1, and EPR spectroscopy indicated that 35 is EPR-silent, both consistent with a ferryl species. Analysis by XAS confirmed the presence of a short FedO distance of 1.687(6) Å, consistent with the ferryl assignment. Resonance Raman spectroscopy on 35 – revealed a band at 735 cm-1 assigned to the n(Fe¼O) (D18O ¼ –32 cm 1). Such a low n(Fe¼O) is unprecedented among previously vibrationally characterized FeIV(O) species, which typically exhibit n(Fe]O) between 800–900 cm-1.195 The highly activated Fe]O bond likely arises from the strong donation from the dithiolato donors, which is expected to weaken the Fe]O bond. Unlike other FeIV(O) complexes,196 35 is unreactive towards OAT reactions with either the internal thiolate donors, or exogenous O-atom acceptors (e.g., phosphines, sulfides). However, 35 reacts readily with H-atom donors containing OdH bonds, including 2,2,6,6- III tetramethylpiperidin-1-ol (TEMPOH) derivatives and phenols, to produce the expected Fe (OH)(Me3TACN)(S2SiMe2)(36). Both –1 –1 III EPR and Mössbauer spectroscopy (d ¼ 0.50 mm s , │DEQ│ ¼ 1.09 mm s ) corroboratea 36 as an Fe (OH) complex, and the XAS data were consistent with the expected geometry, showing an elongation of the FedO bond to 1.907(12) Å which is expected following HAT to 35. In addition, 36 could be independently synthesized by one-electron outer-sphere oxidation and addition of - OH to 33. Complex 33 is a rare example of an iron(II) complex that activates O2 to produce a series of detectable Fe/oxygen intermediates (Scheme 22). This work also shows that each of these intermediates can be formed in the presence of cis-ligated thiolate donors.171

6.3 Reduced O2 Surrogates A number of FeIV(O) complexes have been prepared from O-atom transfer reagents (e.g., ArIO) in the absence of sulfur ligands.197 The ligand in Scheme 23 incorporates a thioether moiety and a potential amide, H-bond donor, and was employed to generate a IV II amide rare sulfur-ligated Fe (O) species. Addition of iodosylbenzene (PhIO) to the low-spin ferrous complex [Fe (N3Py SR)](BF4)2 IV IV amide (37)at−40 C resulted in the formation of an Fe (O) complex, [Fe (O)(N3Py SR)](BF4)2 (38). Mössbauer spectroscopy –1 –1 revealed a quadrupole doublet with d ¼ 0.04 mm s and │DEQ│ ¼ 0.80 mm s , and the low isomer shift observed is typical of FeIV(O) complexes.197 Complex 38 is stable at −40 C, and no sulfur oxidation is seen. However, addition of the exogenous sulfur substrate, thioanisole (PhSMe) results in the rapid reformation of the FeII complex 37 (82% yield) and PhS(O)Me (80% yield). The

Scheme 23 34 Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes same reaction could be performed with the FeII complex as catalyst, leading to 40 turnovers at −40 C. In contrast, addition of the stronger oxidant meta-chloroperoxybenzoic acid (mCPBA), results in direct attack of the thioether ligand to give the sulfoxide II amide 2+ complex [Fe (N3Py S(O)R)] (BF4)2 (39). Taken together these observations indicate that there is a barrier to direct intramo- lecular O-atom transfer from the ferryl to the coordinated sulfur ligand, but not to exogenous thioether substrates. The origins of this inter- versus intramolecular discrimination were not determined. II t Reaction of mCPBA and [Fe (TMCS)](PF6)(40) in the presence of a base, KO Bu, at −40 C produces the thiolate-ligated IV IV Fe (O) complex, [Fe (O)(TMCS)](PF6)(41). This species gave near-infrared bands at 850 nm and 1050 nm, which are charac- IV –1 –1 teristic of nonheme Fe (O) species. Mössbauer spectroscopy revealed parameters of d ¼ 0.19 mm s and |DEQ | ¼ 0.22 mm s , and variable field Mössbauer studies confirmed the expected integer-spin nature of 41. X-ray absorption spectroscopy and DFT studies suggested that 41 had the expected Fe(O)(N4S) coordination sphere, with the sulfur ligated trans to the oxo ligand. IV IV 2+ Interestingly, in contrast to the non-thiolate ligated Fe (O) complex, [Fe (O)(TMC)(CH3CN)] , which reacts with the O-atom acceptor PPh3 to generate OPPh3 and the two electron reduced ferrous complex, 41 is inert towards PPh3. However, 41 is a reactive H-atom abstractor, reacting with 9,10-dihydroanthracene (DHA) to produce a new species distinct from the FeII starting material by III UV-vis spectroscopy (lmax ¼ 514 nm). The authors hypothesized that the expected one electron reduced Fe (OH) product was formed on the basis of mass spectrometry analysis, which showed a peak consistent with [FeIII(TMCS)(OMe)]+. In contrast, [FeIV(O) + (TMC)(CH3CN)] , which does not contain a thiolate ligand, reacts extremely slowly with DHA (Fig. 25). II When [Fe (TMCS)](PF6) is reacted with mCPBA in the absence of a base, a new species is formed. The new species 42 is an II II O-bound Fe (sulfinate) complex, [Fe (TMCSO2)](PF6) based on UV-vis, Mössbauer, and X-ray absorption spectroscopies and mass 198 IV IV spectrometry (Fig. 26). Further mCPBA treatment results in the formation of an Fe (O) complex, [Fe (O)(TMCSO2)](PF6)(43).

IV + IV 2+ Fig. 25 Comparison of reactivity between [Fe (O)(TMCS)] and [Fe (O)(TMC)(CH3CN)] .

II Fig. 26 Reactivity of [Fe (TMCS)](PF6) with mCPBA in the absence of base and Mössbauer spectra following addition of (A) 0 equiv, (B) 1 equiv, (C) 2 equiv, (D) 3 II equiv of mCPBA to [Fe (TMCS)](PF6). Reprinted with permission from reference McDonald, A. R.; Bukowski, M. R.; Farquhar, E. R.; Jackson, T. A.; Koehntop, K. D.; Seo, M. S.; De Hont, R. F.; Stubna, A.; Halfen, J. A.; Münck, E.; Nam, W.; Que, L., Jr. J. Am. Chem. Soc. 2010, 132, 17118–17129. Copyright 2010 American Chemical Society. Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes 35

Complex 43 exhibits a low intensity band at 830 nm, characteristic of nonheme FeIV(O) (S ¼ 1) complexes, and was characterized – – by resonance Raman spectroscopy, exhibiting n(FedO) ¼ 831 cm 1, and Mössbauer spectroscopy exhibiting d ¼ 0.19 mm s 1 and –1 IV |DEQ | ¼ 1.28 mm s . EXAFS analysis revealed a short Fe–O distance of 1.64 Å, expected for an Fe (O) complex. Unlike the thiolate ligated 41, complex 43 was able to transfer an O atom to PPh3. Complex 43 is also able to abstract an H-atom from anthracene at a rate comparable to that of 41. The differences in reactivity for 41 and 43 are not obvious. The formation of 41 versus 43 based on the presence of base suggests that Fe versus S oxygenation is dependent on the presence of a proton. Addition of acid to 41 results in formation of a new FeIV species in 38% yield, in which it was proposed that the thiolate is protonated and no longer bound to the Fe center. It was further proposed that intermolecular O-atom transfer to the thiol results in the formation of 42. Another example of sulfur-ligated iron reacting with strong O-atom donors comes from the reaction of the dithiolate-ligated III Me2 III III 2 Me2 Me2 complex [Fe (S2 N3(Pr,Pr))](PF6)(30), giving the Fe (sulfenate) complex [Fe (Z -S O)(S N3(Pr,Pr))](PF6)(32) as shown 199 III Me2 in Fig. 27. In contrast, reaction of PhIO with the azide-bound, 6-coordinate complex [Fe (S2 N3(Pr,Pr))N3] resulted in no reaction, suggesting that S-oxygenation occurred via a metal mediated process rather than direct sulfur attack. Running the reaction of PhIO with 30 at –73 C resulted in the formation of a new intermediate (lmax ¼ 677 nm), which was hypothesized to be either FeV(O) or an iron(III)-iodosylarene adduct. The reactivity of 30 was compared with that of the more electron-rich analogue III Me2 Me amide containing carboxamido donors, [Et4N][Fe S2 N N2 (Pr,Pr)] (44)(Fig. 27). In contrast to 30, 44 does not readily bind a sixth ligand, and thus undergoes no reaction with O-atom donors. However, DFT calculations comparing 30 and 44 indicate that significantly more electron density is located on the S donors in 44, indicating that they should be more easily oxidized. These results suggest that oxidation of a sulfur donor, as seen for 30, occurs via the formation of an Fe-based oxidant. II Me2 + Addition of KO2 to a solution of [Fe (S N4(tren))] (20) in the presence of trace water at ambient temperature results in the III Me2 2+ formation of [Fe S N4(tren)(MeCN)] , which was characterized crystallographically. The formation of this ferric complex suggested that 20 is able to reduce superoxide.200 When the same reaction is carried out in MeOH at −90 C, a transient intermediate III Me2 + 201 45 is formed, but decays to [Fe S N4(tren)(OMe)] (46) within 10 min. Complex 45 (lmax ¼ 452 nm) gives an EPR spectrum with g ¼ [2.14, 1.97], indicative of a low-spin ferric center. Resonance Raman spectroscopy showed an isotopically sensitive Fermi –1 –1 18 doublet (788, 781 cm ) that collapsed to a single peak at 784 cm with MeOD, and substitution of K O2 resulted in a down-shift –1 III Me2 + to 753 cm . These vibrational data are consistent with a peroxo ligand, and 45 was assigned as low-spin [Fe S N4(tren)(OOH)] (Fig. 28). Further evidence for this assignment came from XAS studies, which indicated that 45 was a 6-coordinate Fe(N4SO) complex, consistent with an Fe–O(peroxo) bond length of 1.86(3) Å. Decay of 45 proceeds to give an FeIII(OMe) complex.202 No further substrate reactivity for 45 was reported. II + The ferrous complex [Fe (cyclam-PrS)] (47) with an open coordination site trans to an alkylthiolate donor, reacts with KO2 in III + the presence of a proton donor (MeOH) to produce a metastable intermediate [Fe (cyclam-PrS)(OOH)] (48)(lmax ¼ 530 nm) (Scheme 24).175 EPR analysis gave g ¼ [7.72, 5.40, 4.15], indicative of a high-spin ferric center. Resonance Raman spectroscopy on –1 –1 –1 18 48 reveals a n(OdO) band at 891 cm and n(FedO) at 419 cm , which downshift to 856 and 400 cm , respectively, with K O2.

III III Me2 III Me2 Me amide Fig. 27 (A) Formation of Fe (sulfenate) from [Fe (S2 N3(Pr,Pr))]PF6. (B) Reaction of [Et4N][Fe S2 N N2 (Pr,Pr)] with PhIO. (C) Crystal structure of 32. Reprinted with permission from reference Villar-Acevedo, G.; Lugo-Mas, P.; Blakely, M. N.; Rees, J. A.; Ganas, A. S.; Hanada, E. M.; Kaminsky, W.; Kovacs, J. A. J. Am. Chem. Soc. 2017, 139, 119–129. Copyright 2017 American Chemical Society. 36 Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes

(B) (A) 100 + + 80 + OH O OMe 753 60 NH2 N KO2 N NH Decay N NH III 2 III 2 40 FeII Fe Fe % Trans. N SSMeOH N N S 20 H2 −90 °C H2 H2 N N N 0 800 790 780 770 760 750 740 730 20 45 46 cm−1

II Me2 + 16 18 Fig. 28 (A) Reactivity of [Fe (S N4(tren))] with KO2 in MeOH. (B) IR spectrum of 45 prepared from K O2 (dashed line) and K O2 (solid line). Reprinted with permission from reference Shearer, J.; Scarrow, R. C.; Kovacs, J. A. J. Am. Chem. Soc. 2002, 124, 11709–11717. Copyright 2002 American Chemical Society.

Scheme 24

III + Addition of HOAc results in the release of H2O2 and formation of [Fe (cyclam-PrS)(OAc)] , which could be reduced to 47 using II Co (Cp)2, indicating that 47 should perform catalytic superoxide reduction. II 192 Addition of excess (>50 equiv) KO2 to a solution of [Fe (TMCS)](PF6)at−90 C results in the formation of a new species 49, II which decayed over time to re-form [Fe (TMCS)](PF6), suggesting a possible equilibrium of superoxide binding. EPR and Mössbauer spectroscopy were consistent with the formation of an S ¼ 5/2 ferric species, giving g ¼ 7.9, 5.75, and d ¼ 0.71 mm –1 –1 s , DEQ ¼ −1.9 mm s , respectively. XAS analysis was consistent with an Fe(N4SO) coordination environment, leading to the III - assignment of 49 as an end-on peroxide anion complex (Scheme 25), [Fe (O2)(TMCS)]. Addition of excess acid resulted in the formation of a new species tentatively assigned as the hydroperoxo complex, [FeIII(OOH)(TMCS)]+. However, no further charac- terization was carried out. Complex 49 was shown to be unreactive towards CdH bonds, but reacted with electrophiles such as menadione to produce the corresponding epoxide, and 2-phenylpropionaldehyde to produce acetophenone and formate.

Scheme 25

The tetraazamacrocycle complexes with tethered alkyl thiolate donors have been shown to stabilize Fe/oxygen intermediates. To access a series of FeII(thiolate) complexes in which the thiolate donor strength can be rationally tuned as a function of its electronic properties, a series of ferrous complexes coordinated to the tetraazamacrocycle, [15]aneN4, were prepared with different 203 II t axial thiolate donors (Fig. 29). The reaction of [Fe ([15]aneN4)(SPh)]BF4 (50a) with BuOOH or cumenyl hydroperoxide III (CmOOH) in CH2Cl2 at −78 C resulted in the formation of the low-spin alkylperoxo complexes [Fe ([15]aneN4)(SPh)(OOR)] 177 −1 −1 BF4 (51a,R¼ tBu, 51f:R¼ Cm). These complexes exhibited UV-vis spectral features at lmax ¼ 526 nm (2150 M cm ) and lmax − − ¼ 527 nm (1650 M 1 cm 1) for 51a and 51f, respectively, which were assigned to alkylperoxo-to-Fe(III) ligand to metal charge transfer bands. These complexes were further characterized by EPR spectroscopy, giving g ¼ 2.20 and 1.97, and resonance Raman −1 t 16 t 18 −1 spectroscopy, which showed n(OdO) features 800 cm (Dn( Bu O2H– Bu O2H) ¼ −46 cm for 51a) and n(FedO) 615 −1 t 16 t 18 −1 cm (Dn( Bu O2H– Bu O2H) ¼ –28 cm for 51a). The Fe(OOR) complexes for each of the thiolate donors shown in Fig. 29 − − exhibited UV-vis features between 500 and 550 nm, n(OdO) features 800 cm 1, and n(FedO) between 600 and 635 cm 1.203 Further characterization by XAS in conjunction with density functional theory calculations were in accordance with the assignments of these species as low-spin alkyl peroxo complexes.204 Interestingly, the n(FedO) increases as the electron donating ability Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes 37

III II Fig. 29 (A) Formation of [Fe ([15]aneN4)(SPh)(OOR)]BF4 complexes. (B) Plot of n(OdO) and n(FedO) versus redox potential for a series of Fe (thiolate) complexes.

decreases, suggesting a weakening of the FedO bond with increasing electron density from the axial donor, with a nearly linear trend observed in the plot of n(FedO) versus the redox potential of the starting ferrous complex (Fig. 29). However, the n(OdO) remains relatively invariant across the series of complexes. These results indicate that the trans thiolate donor can tune the FedO bond strength without significantly altering the OdO bond strength. The rates of decay of these complexes were measured and revealed that the rates increased as the FedO bond strength decreased. These observations are consistent with FedO bond cleavage being involved in the decay process. However, attempts to identify the decomposition products were unsuccessful.204 The decay of the series was accelerated in the presence of acids or aldehydes, suggesting these complexes may exhibit nucleophilic character. However, no products were identified. No reactivity with weak O-atom acceptors or H-atom donors was observed. II The structurally related Fe complexes 37 and 52, with and without a potential H-bonding amide group, react with H2O2 or tBuOOH to generate a series of FeIII(OOR) (S ¼ ½) complexes (53–54)(R¼ tBu, H), characterized by resonance Raman, UV-vis, and EPR spectroscopies (Fig. 30).178 The presence of the H-bonding moiety shifted the UV-vis feature associated with the alkylperoxo-to- Fe(III) ligand to metal charge transfer bands, but only minor changes in the n(OdO) and n(FedO) vibrational modes were observed, indicating that H-bonding to the peroxo unit does not significantly perturb the Fe/O2 bond strengths. Interestingly, a significant decrease in n(OdO) and increase in n(FedO) were observed when compared with the all-nitrogen-ligated [FeIII(N4Py) (OOH)]2+, suggesting that the cis-sulfur ligation weakens the OdO bond while strengthening the FedO bond.205

6.4 Models of Thiolate-Ligated Nonheme Iron Enzymes with Other 3d Metals Though the structure and reactivity of Fe–thiolate complexes provide the most direct comparison with enzymatic reactivity, analogous studies using other metals are also germane to our understanding of how O2 can be activated at a metal center in the

Fig. 30 (A) Reactions of [FeII(N3PySR)]2+ complexes with ROOH (R]H, tBu). (B) Resonance Raman spectra of FeIII(OOtBu) complexes. Reproduced from reference Widger, L. R.; Jiang, Y.; McQuilken, A. C.; Yang, T.; Siegler, M. A.; Matsumura, H.; Moënne-Loccoz, P.; Kumar, D.; de Visser, S. P.; Goldberg, D. P. Dalton Trans. 2014, 43, 7522–7532, with permission from The Royal Society of Chemistry. 38 Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes

Scheme 26 presence of thiolate donors. Studies using Co as a surrogate for Fe, in particular, have a long history in the field of bioinorganic 206 chemistry in helping to understand fundamental aspects of O2 binding. III + The crystallographically characterized Cr(superoxo) species, [Cr (O2)(TMC)(Cl)] (55-O2) was shown to undergo a single O-atom transfer to thioanisole to generate [CrIV(O)(TMC)(Cl)]+ (55-O)(Scheme 26). This report provided evidence that a metal- superoxo species could directly oxidize a sulfur substrate, as proposed for CDO. II III A series of Mn –thiolate complexes react with O2 to generate unsupported Mn oxo-bridged dimers, which were characterized 207 II Me2 crystallographically. Reaction of [Mn (S N4(6-Me-DPEN))](BPh4)(56) with O2 at low temperature (−80 C) produced a III binuclear Mn2 -trans-1,2-peroxo complex (57) that was characterized structurally by XRD (Fig. 31), and, upon warmup, led to – – – formation of the oxo-bridged dimer.208 This complex gave n(OdO) ¼ 819 cm 1 (D18O ¼ −47 cm 1) and n(MndO) ¼ 611 cm 1 – (D18O ¼ −25 cm 1). Magnetic susceptibility measurements indicated that the complex is uncoupled (J ¼ 0cm-1). Prior to formation of the peroxo species, a transient intermediate with lmax ¼ 515 nm was observed by stopped-flow UV-vis spectroscopy. This III intermediate was proposed to be a Mn(superoxo) species, which rapidly converted to the Mn2 (m-O2) complex. It was proposed that III IV the formation of the Mn2 (mdO) complex occurred via homolytic OdO bond cleavage of 57 to generate a Mn (O) species, which III then goes on to give the final Mn2 (mdO) complex. This hypothesis is supported from the observation that PPh3 and 2,4-di-tert- butylphenol are both oxidized in the reaction of 56 with O2. However, there was no direct evidence for the formation of any high- valent MnIV(O) species. A recent study using a more electron-rich and less sterically bulky ligand derivative found that several other III •− IV III IV intermediates, assigned as Mn (O2 ), Mn2 (O)2, and Mn Mn (O)(OH) species, could be observed by stopped-flow UV-vis spectroscopy. A detailed kinetic analysis of a series of related MnII complexes revealed that metal Lewis acidity as well as ligand steric properties play key roles in the binding of dioxygen and subsequent OdO bond cleavage steps.209 I Reaction of the tris(thiolato)-ligated Mn complex [PPN][Mn(CO)3(P(C6H3-3-R-2-S)2(C6H3-3-R-2-SH))] (R]SiMe3)(58) with IV TMS excess O2 leads to a mononuclear S ¼ 3/2 side-on Mn(O2) adduct, [PPN][Mn ( PS3)(O2)] (59), that was structurally characterized by XRD, which indicated an OdO bond length of 1.379(3) Å. Vibrational analysis by infrared spectroscopy gave – – n(OdO) ¼ 903 cm 1 (D18O ¼ –42 cm 1), which is in the range typically seen for peroxo species, leading the authors to formulate 59 as a MnIV(peroxo) species.210 Protonation of this complex is proposed to lead to the formation of a putative MnIV(OOH) complex that can rapidly release hydroperoxyl radical, which could be scavenged by PPh3. However, no direct evidence of the MnIV(OOH) complex was obtained.211 Addition of 59 to the MnII complex [PPN][MnII(TMSPS3)(DABCO)] (60) (DABCO ¼ 1,4- III diazabicyclo[2.2.2]octane) results in the formation of a Mn2 (mdO) complex (61)(Scheme 27), which was proposed to form via an III 212 intermediate Mn2 (O2) dimer, though this intermediate could not be detected. II II + 2− 0 0 0 0 The Mn dimer, [Mn2(LS)(LSH)] (62) (where LS ¼2,2 -(2,2 -bipyridine-6,6 -diyl)bis(1,1 -diphenylethanethiolate)) contains III a bound thiol moiety and reacts with air to perform the four-electron reduction of O2, producing a dimeric Mn2 (m–OH) complex (63)(Scheme 28). It was proposed that this intermediate is formed via an undetected dimeric peroxo intermediate prior to eventual formation of 63. In contrast, addition of O2 to 62 in the presence of an acid source leads to two-electron reduction of dioxygen and

Fig. 31 Crystal structure of complex 57. Reprinted with permission from reference Coggins, M. K.; Sun, X.; Kwak, Y.; Solomon, E. I.; Rybak-Akimova, E.; Kovacs, J. A. J. Am. Chem. Soc. 2013, 135, 5631–5640. Copyright 2013 American Chemical Society. Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes 39

Scheme 27

Scheme 28

213 release of H2O2. In the presence of an exogenous reductant, 62 could perform the reduction of O2 to H2O2 catalytically. II IV Deprotonation of 62 leads to the formation of [Mn2(LS)2](64), which reacts with O2 to produce the high-valent Mn2 (O)2 dimer IV (65). Protonation of this species gives Mn2 (O)(OH) (66). It was found that addition of 62–66 leads to a comproportionation 214 reaction to generate 63, implicating 66 as an intermediate in the four-electron reduction of O2 by 62. II II Me2 III The Co complex [Co (S N4(tren))](PF6)(67) was shown to bind O2, generating a diamagnetic, binuclear Co2 -trans-1,2- peroxo complex (68) that was characterized crystallographically (Scheme 29). Complex 68 exhibited a CodO and OdO bond lengths of 1.899(2) Å and 1.482(4) Å, respectively. The OdO bond is slightly longer than a Mn2(peroxo) species, 57, with a similar ligand set. DFT calculations suggested that the long OdO bond may arise from the lack of donation from the p(OdO) orbital into the filled cobalt d orbitals. II R2 The Tp-ligated Co complexes, [Co(Tp )(CysOEt)] (R]Ph (69), Me (70)), were prepared and their reactivity with O2 was III examined (Scheme 30). Addition of O2 to 69 resulted in no reaction, possibly due to the steric profile of the ligand, or high Co / II Me2 Co redox potentials. Addition of O2 to 70 at −70 C leads to the formation of [Co(O2)(Tp )(CysOEt)] (71), which was identified III •− 59 −1 as a Co (O2 ) complex by its characteristic EPR signature with small Co hyperfine coupling, and a n(OdO) mode at 1152 cm − (D18O ¼ 61 cm 1) from resonance Raman spectroscopy. In contrast to the analogous Fe complex, no S-oxygenation is observed and II instead, release of O2 and reformation of the Co starting complex is seen upon warmup. II II The Co (silanedithiolate) (S ¼ 3/2) complex Co (Me3TACN)(S2SiMe2)(72), which is the Co analogue of the Fe complex, 33, 215 was prepared in order to stabilize a Co(superoxo) species bound to thiolate donors. Addition of excess O2(g), or one equivalent of O2 in an O2-saturated 2-MeTHF solution to 72 at −80 C resulted in the formation of 73, which exhibits UV-vis spectral features at 40 Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes

Scheme 29

Scheme 30

340, 360, 455, and 555 nm (Fig. 32). Complex 73 is stable at −80 C, and does not revert back to 72 upon sparging with argon gas or under reduced pressure. Characterization by EPR spectroscopy gave g 2.11, 2.02, 1.98 and A(59Co) ¼ 30 G, typical of an S ¼ 1/2 − cobalt superoxo complex. The assignment of 73 was confirmed by resonance Raman spectroscopy, with a peak at 1133 cm 1 that −1 18 downshifts to 1070 cm upon O2 substitution, which was in line with the O–O stretch for a superoxide complex. Additional O2- -1 18 16 −1 –1 18 16 –1 sensitive features at 520 cm (Dn( O2– O2) ¼ –29 cm ) and 367 cm (Dn( O2– O2) ¼ –9cm ) were assigned to CodO stretching and Co–O2 bending modes, respectively. Analysis by EXAFS was consistent with the proposed Co(O2)(Me3TACN) (S2SiMe2) structure, and DFT calculations were in accordance with the experimental data. Interestingly, analysis of the Co K-edge XAS spectrum revealed that the pre-edge and rising edge energies for 72 and 73 are nearly identical. These observations suggested that upon binding of O2, redox chemistry is not occurring exclusively at the Co center. DFT calculations on 73 revealed significant hole character on the thiolate donors, suggesting that the entire [CoS2] unit is acting as the redox active moiety and providing the electron for reducing O2 to superoxide. The reactivity of 73 was tested with a variety of O-atom acceptors, including phosphines, sulfides, and alkylthiolates, exhibiting no reactivity with these compounds. In addition, 73 was stable against intramolecular attack of the bound thiolate donors, indicating that the superoxide moiety in 73 is relatively inert toward sulfur oxidation. In contrast, reaction of 73 with the H-atom donor 4-methoxy-2,2,6,6-tetramethylpiperidin-1-ol (4-OMe-TEMPOH) or the un-substituted analogue TEMPOH, resulted in rapid decay of the features associated with 73 and formation of the corresponding nitroxyl radicals (Fig. 32). A KIE of 8.8 for the { – { reaction with TEMPOH(D) was obtained at −105 C. An Eyring analysis gave activation parameters DH ¼ 3.6 kcal mol 1 and DS ¼ – – −46.2 cal mol 1 K 1, indicating a highly ordered transition state. The bond dissociation free energy (BDFE) of the putative formed

Co(OOdSH) bond was estimated from the reactivity of 73 with separated acid/reductant pairs of various strengths (e.g., Et3NH/ –1 –1 Fe(Cp )2 effective BDFE ¼ 70 kcal mol ). The reactivity pattern gave an upper limit BDFE of 70 kcal mol for the hydroperoxo – OdH bond. This estimate was matched nicely with a DFT calculated OdH bond strength of 67 kcal mol 1. These results indicate that 73 is only a weak H-atom abstractor, but lends support to the feasibility of a metal(superoxo) complex preferentially oxidizing an OdH bond over a sulfur ligand, as proposed in the mechanism of EgtB/OvoA.

7 Outlook and Future Work

The burgeoning class of O2 activating nonheme iron enzymes containing thiolate ligation has emerged as an important and mechanistically complex group of enzymes. These enzymes, IPNS, the thiol dioxygenases, the sulfoxide synthases, and the persulfide dioxygenases, are all functionally unique yet share structural and mechanistic similarities. However, the chemistry carried out by these enzymes following the binding of O2 diverges markedly, involving either H-atom abstraction, S-oxygenation, or possibly both, via poorly understood mechanisms. Elucidating these different mechanisms and trapping relevant intermediates remains a challenging and important goal for future mechanistic studies. Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes 41

Fig. 32 (A) Formation and reactivity of Co(O2)(Me3TACN)(S2SiMe2). (B) UV-vis, EPR, and (C) X-ray absorption (XANES) spectra for Co(O2)(Me3TACN)(S2SiMe2). Black lines in B and C corresponds to data for 72 and red lines correspond to data for 73. Adapted with permission from reference Gordon, J. B.; Vilbert, A. C.; Siegler, M. A.; Lancaster, K. M.; Moënne-Loccoz, P.; Goldberg, D. P. J. Am. Chem. Soc. 2019, 141, 3641–3653. Copyright 2019 American Chemical Society.

A key question surrounding these mechanisms is why and how their reactivities diverge. Though the mechanism for IPNS is fairly well established, it is unclear how the iron center in IPNS preferentially abstracts the Cys b H-atoms over S-oxygenation of the bound sulfur ligand. Conversely, it is unclear why the iron/oxygen pathway in CDO does not lead to attack of the Cys b H-atom of the chelated Cys substrate. Perhaps one of the most interesting mechanistic observations for these enzymes is that their reactivities can be interconverted. For example, addition of an ACV substrate analogue (ACOmV) to IPNS leads to S-oxygenation, generating a sulfenate species, which is a proposed intermediate in CDO.30 The sulfoxide synthases EgtB and OvoA can be converted into thiol dioxygenases with appropriate mutations or substrates.216 The persulfide dioxygenase ETHE1 was shown to undergoes single turnover S-dioxygenation reactivity analogous to the thiol dioxygenases.154 These observations suggest that these enzymes are all mechanistically related, and studies of each of these enzymes can lend important information for the entire class. As structural data are obtained for the enzymes with and without substrates bound, our understanding of sulfur-ligated nonheme iron enzymes will be refined, and more mechanistic studies guided by structure can be carried out. Though several of these sulfur-ligated enzymes have been known for decades, the recent discovery of the sulfoxide synthases121,122,124 as well as new thiol dioxygenases such as MSDO43 and plant cysteine oxidase,45 suggests that there are many fascinating new related enzymes to be discovered. Many of these intriguing questions can be addressed by examination of synthetic model complexes. The study of model complexes provides fundamental spectroscopic signatures of Fe/oxygen intermediates in the presence of sulfur donors, which can be essential in the assignment of intermediates observed in enzymatic studies. It is apparent from studies with well-defined synthetic metal–thiolate complexes that the key factors controlling the preference for S-oxygenation or H-atom abstraction are not yet understood. However, in the last two decades, we have seen interesting examples of both reactivity patterns. The ability to exert precise control over the coordination sphere of synthetic Fe–thiolate complexes provides significant promise for understanding the guiding principles that determine these two pathways. Ultimately, the insights developed from studies of these enzymes and synthetic model complexes can be directed towards the rational design of new synthetic catalysts that can bind and activate O2 for the controlled, selective oxidation of organic substrates. 42 Sulfur-Ligated, Oxidative Nonheme Iron Enzymes and Related Complexes

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