Sulfur-Ligated, Oxidative Nonheme Iron Enzymes 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 Cysteine Dioxygenase 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 oxygenases 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 catalysis 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 substrate 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 enzyme, isopenicillin N synthase (IPNS), which uses a mononuclear iron cofactor 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 active site 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 product 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 oxygenase 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)