Spectroscopic and computational studies of nitrile hydratase: insights into geometric and electronic structure and the mechanism of amide synthesis

Light, Kenneth M.; Yamanaka, Y.; Odaka, Masafumi; Solomon, Edward I.

doi:10.1039/c5sc02012c

Cited by 20 publications

(58 citation statements)

References 38 publications

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“…1C) and thus strongly support the mechanism involving the enzymebased oxygen nucleophile in the form of a sulfenate nucleophile (Fig. 1B), which is supported by recent computational studies suggesting the sulfenato nucleophile mechanism (4,5). This work provides the first direct evidence from the solution phase that a protein-based nucleophilic oxygen is the source of the carboxamido oxygen in product amides of FIGURE 7.…”

Section: Figure 2 Analysis Of Toyj Mass Spectrometry Data For Incorpsupporting

confidence: 63%

A Protein-derived Oxygen Is the Source of the Amide Oxygen of Nitrile Hydratases

Nelp

Song

Wysocki

et al. 2016

Journal of Biological Chemistry

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Nitrile hydratase metalloenzymes are unique and important biocatalysts that are used industrially to produce high value amides from their corresponding nitriles. After more than three decades since their discovery, the mechanism of this class of enzymes is becoming clear with evidence from multiple recent studies that the cysteine-derived sulfenato ligand of the active site metal serves as the nucleophile that initially attacks the nitrile. Herein we describe the first direct evidence from solution phase catalysis that the source of the product carboxamido oxygen is the protein. Using 18 O-labeled water under single turnover conditions and native high resolution protein mass spectrometry, we show that the incorporation of labeled oxygen into both product and protein is turnover-dependent and that only a single oxygen is exchanged into the protein even under multiple turnover conditions, lending significant support to proposals that the post-translationally modified sulfenato group serves as the nucleophile to initiate hydration of nitriles. Nitrile hydratases (NHases)2 catalyze the formation of amides from nitriles in aqueous, pH-neutral environments without any carboxylic acid byproducts (1, 2). These bacterial enzymes utilize trivalent iron or cobalt metal cations in this non-redox reaction, and uniquely, these metal ions are ligated directly to the protein backbone through amidate ligands and three cysteine sulfur ligands, of which two are post-translationally modified to the sulfinic and sulfenic acid ( Fig. 1A) (3). The mechanism of hydration is slowly emerging through multiple studies and appears to involve the sulfenic acid oxygen as the nucleophile. This sulfenate oxygen is proposed to initially attack the metal-bound nitrile carbon atom, creating a cyclic intermediate that is then resolved through attack at the sulfenic sulfur either directly by water or from the neighboring axial thiolato sulfur (Fig. 1B) (4 -6). Alternative mechanisms have also found support from time-resolved crystallographic and kinetic studies that involve an activated water molecule attacking the metal-bound nitrile (Fig. 1C) (7, 8).Early studies on sulfenato ligands in cobalt complexes demonstrated the nucleophilicity of the sulfenato oxygen (9, 10), and recently, this same characteristic was observed in NHases wherein the sulfenato oxygen reacted with electrophilic boronic acids (11). Quite tellingly, the activities of NHase, the related thiocyanate hydrolase, and NHase mimics were found to depend on the presence of these sulfenato ligands where oxidation to the sulfinate abrogated activity (12-14).To find direct evidence of either a protein-based or a solventbased nucleophile, we became interested in the observations made by Heinrich et al. (14) with an iron NHase mimic bearing two sulfenato ligands that is capable of hydrating nitriles with up to 50 turnovers. They discovered that labeled oxygen from solvent water was exchanged into one of these sulfenato groups only if the nitrile hydration reaction occurred. This provides a...

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Section: Figure 2 Analysis Of Toyj Mass Spectrometry Data For Incorpsupporting

confidence: 63%

A Protein-derived Oxygen Is the Source of the Amide Oxygen of Nitrile Hydratases

Nelp

Song

Wysocki

et al. 2016

Journal of Biological Chemistry

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“…This form of the enzyme exhibits maximal catalytic activity, is reversibly inhibited by butyric acid, and is irreversibly deactivated by exposure to oxygen. A species with g values very similar to those of N b was assigned to the active “NHaseAq” species of Rh NHase-N771 16 and was also observed in Pc NHase-23. 13 Taylor/DFT calculations based on the active enzyme model again reproduced the g tensor to unprecedented precision and were clearly very distinct from those of other structural models (Table 1).…”

Section: Discussionmentioning

confidence: 78%

Multiple States of Nitrile Hydratase from Rhodococcus equi TG328-2: Structural and Mechanistic Insights from Electron Paramagnetic Resonance and Density Functional Theory Studies

et al. 2017

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Iron-type nitrile hydratases (NHases) contain an Fe(III) ion coordinated in a characteristic “claw setting” by an axial cysteine thiolate, two equatorial peptide nitrogens, the sulfur atoms of equatorial cysteine-sulfenic and cysteine-sulfinic acids, and an axial water/hydroxyl moiety. The cysteine-sulfenic acid is susceptible to oxidation, and the enzyme is traditionally prepared using butyric acid as an oxidative protectant. The as-prepared enzyme exhibits a complex electron paramagnetic resonance (EPR) spectrum due to multiple low-spin (S = 1/2) Fe(III) species. Four distinct signals can be assigned to the resting active state, the active state bound to butyric acid, an oxidized Fe(III)–bis(sulfinic acid) form, and an oxidized complex with butyric acid. A combination of comparison with earlier work, development of methods to elicit individual signals, and design and application of a novel density functional theory method for reproducing g tensors to unprecedentedly high precision was used to assign the signals. These species account for the previously reported EPR spectra from Fe-NHases, including spectra observed upon addition of substrates. Completely new EPR signals were observed upon addition of inhibitory boronic acids, and the distinctive g1 features of these signals were replicated in the steady state with the slow substrate acetonitrile. This latter signal constitutes the first EPR signal from a catalytic intermediate of NHase and is assigned to a key intermediate in the proposed catalytic cycle. Earlier, apparently contradictory, electron nuclear double resonance reports are reconsidered in the context of this work.

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“…A similar reaction sequence was recently observed to convert a sulfur-bound substrate analogue of isopenicillin-N-synthase to an Fe II ((η 2 -SO), 132 and may be the mechanism by which the catalytically important sulfenate is inserted into the Fe-NHase active site. 27 …”

Section: Resultsmentioning

confidence: 99%

“…25, 26 They are also required for enzyme activity in some cases (e.g, nitrile hydratase (NHase), 27–31 NADH peroxidase, 32 and peroxiredoxins 33 ). Nitrile hydratases are a class of thiolate-ligated non-heme iron enzymes for which cysteinate oxygenation is intimately tied to function.…”

Section: Introductionmentioning

confidence: 99%

Metal-Assisted Oxo Atom Addition to an Fe(III) Thiolate

et al. 2016

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Cysteinate oxygenation is intimately tied to the function of both cysteine dioxygenases (CDOs) and nitrile hydratases (NHases), and yet the mechanisms by which sulfurs are oxidized by these enzymes are unknown, in part because intermediates have yet to be observed. Herein, we report a five-coordinate bis-thiolate ligated Fe(III) complex, [FeIII(S2Me2N3-(Pr,Pr))]+ (2), that reacts with oxo atom donors (PhIO, IBX-ester, and H2O2) to afford a rare example of a singly oxygenated sulfenate, [FeIII(η2-SMe2O)(SMe2)N3(Pr,Pr)]+ (5), resembling both a proposed intermediate in the CDO catalytic cycle and the essential NHase Fe-S(O)Cys114 proposed to be intimately involved in nitrile hydrolysis. Comparison of the reactivity of 2 with that of a more electron-rich, crystallographically characterized derivative, [FeIIIS2Me2NMeN2amide(Pr,Pr)]− (8), shows that oxo atom donor reactivity correlates with the metal ion’s ability to bind exogenous ligands. Density functional theory calculations suggest that the mechanism of S-oxygenation does not proceed via direct attack at the thiolate sulfurs; the average spin-density on the thiolate sulfurs is approximately the same for 2 and 8, and Mulliken charges on the sulfurs of 8 are roughly twice those of 2, implying that 8 should be more susceptible to sulfur oxidation. Carboxamide-ligated 8 is shown to be unreactive towards oxo atom donors, in contrast to imine-ligated 2. Azide (N3−) is shown to inhibit sulfur oxidation with 2, and a green intermediate is observed, which then slowly converts to sulfenate-ligated 5. This suggests that the mechanism of sulfur oxidation involves initial coordination of the oxo atom donor to the metal ion. Whether the green intermediate is an oxo atom donor adduct, Fe-O═I-Ph, or an Fe(V)═O remains to be determined.

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Spectroscopic and computational studies of nitrile hydratase: insights into geometric and electronic structure and the mechanism of amide synthesis

Cited by 20 publications

References 38 publications

A Protein-derived Oxygen Is the Source of the Amide Oxygen of Nitrile Hydratases

A Protein-derived Oxygen Is the Source of the Amide Oxygen of Nitrile Hydratases

Multiple States of Nitrile Hydratase from Rhodococcus equi TG328-2: Structural and Mechanistic Insights from Electron Paramagnetic Resonance and Density Functional Theory Studies

Metal-Assisted Oxo Atom Addition to an Fe(III) Thiolate

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