Cobalt(III) Complexes with Carboxamido-N and Sulfenato-S or Sulfinato-S Ligands Suggest that a Coordinated Sulfenate-S is Essential for the Catalytic Activity of Nitrile Hydratases

Heinrich, Laurent; Mary-Verla, Aude; Li, Yun; Vaissermann, Jacqueline; Chottard, Jean‐Claude

doi:10.1002/1099-0682(200109)2001:9<2203::aid-ejic2203>3.0.co;2-a

Cited by 64 publications

(106 citation statements)

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“…The main lines of evidence are the following: (i) These modifications were first revealed in the X‐ray structure analysis of N771 NHase and confirmed by mass spectrometric analysis of the active site peptide using Fourier transform ion cyclotron resonance 20. (ii) Using a model complex of Co‐NHase, the sulfenic group has been shown to promote nitrile hydration,33 strongly suggesting its involvement in the NHase‐dependent hydration of nitriles. (iii) Molecules known to react with protein or non‐protein free sulfenic acids were found to inhibit nitrile hydration catalysed by R312 and NI1 Fe‐NHases 34.…”

Section: Discussionmentioning

confidence: 95%

“…Data from recent studies on model complexes support this conclusion. A model complex related to the cobalt‐containing NHase active site, containing two carboxamido nitrogens and two sulfenates, was found to be unstable at acidic pH 33. Finally, the presence of an SO group in the active site of N771 NHase has been discovered only in the mass spectra of the nitrosylated active site peptide collected using Fourier transform ion cyclotron resonance under neutral conditions 20…”

Section: Discussionmentioning

confidence: 99%

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Post‐translational modification of Rhodococcus R312 and Comamonas NI1 nitrile hydratases

et al. 2003

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Nitrile hydratases (NHases) are industrially significant iron- and cobalt-containing enzymes used in the large-scale synthesis of acrylamide. Previous reports have shown that the active site peptides of NHases are post-translationally modified by oxidation of cysteine residues, and that these modifications are essential for catalysis. We report mass spectrometric evidence of the oxidation states of the active site cysteines in the iron coordination spheres of two iron-containing nitrile hydratases, namely R312 NHase from Rhodococcus rhodochrous strain R312 and NI1 NHase from Comamonas testosteroni. At least one of these cysteines is oxidised to a sulfinic acid (SO(2)H) and there is also evidence suggesting an additional oxidation to a sulfenic acid (SOH). This is the first evidence for the presence of these oxidation states for full-length NHases and for Fe-NHases from different microorganisms. The presence of these covalent modifications was confirmed by performing mass spectrometry on the active site peptide of R312 NHase, under native, reduced and carboxymethylated conditions. We also show the nitrosylation of the iron by mass spectrometry, as well as the release of NO by photoirradiation.

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Section: Discussionmentioning

confidence: 95%

Section: Discussionmentioning

confidence: 99%

Post‐translational modification of Rhodococcus R312 and Comamonas NI1 nitrile hydratases

et al. 2003

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“…37 Very few models containing singly oxygenated sulfurs are available, and none of these contain iron. [38][39][40] Only one model has been reported containing both a singly oxygenated and a doubly oxygenated thiolate sulfur in the same molecule. 40 The selective incorporation of an odd number of oxygens is nontrivial, since sulfenates (RS-O(H)) are more reactive than thiolates and readily disproportionate to Herein, the synthesis and characterization of a series of structurally related Fe-NHase model complexes is described, in which the oxidation and protonation states of one of the thiolates are systematically altered.…”

Section: Author Manuscript Author Manuscriptmentioning

confidence: 99%

How Does Single Oxygen Atom Addition Affect the Properties of an Fe−Nitrile Hydratase Analogue? The Compensatory Role of the Unmodified Thiolate

et al. 2006

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Nitrile hydratase (NHase) is one of a growing number of enzymes shown to contain posttranslationally modified cysteine sulfenic acids (Cys-SOH). Cysteine sulfenic acids have been shown to play diverse roles in cellular processes, including transcriptional regulation, signal transduction, and the regulation of oxygen metabolism and oxidative stress responses. The function of the cysteine sulfenic acid coordinated to the iron active site of NHase is unknown. Herein we report the first example of a sulfenate-ligated iron complex, [Fe III (ADIT)(ADIT-O)] + (5), and compare its electronic and magnetic properties with those of structurally related complexes in which the sulfur oxidation state and protonation state have been systematically altered. Oxygen atom addition was found to decrease the unmodified thiolate Fe-S bond length and blue-shift the ligand-to-metal charge-transfer band (without loss of intensity). S K-edge X-ray absorption spectroscopy and density functional theory calculations show that, although the modified RS-O − fragment is incapable of forming a π bond with the Fe III center, the unmodified thiolate compensates for this loss of π bonding by increasing its covalent bond strength. The redox potential shifts only slightly (75 mV), and the magnetic properties are not affected (the S = ½ spin state is maintained). The coordinated sulfenate S-O bond is activated and fairly polarized (S + -O − ). Addition of strong acids at low temperatures results in the reversible protonation of sulfenateligated 5. An X-ray structure demonstrates that Zn 2+ binds to the sulfenate oxygen to afford [Fe III HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript unmodified NHase thiolate, involving its ability to "tune" the electronics in response to protonation of the sulfenate (RS-O − ) oxygen and/or substrate binding, is discussed.Nitrile hydratases (NHases) are non-heme iron enzymes that convert nitriles to less toxic amides cleanly, and rapidly, under mild conditions. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16] It is unusual for a hydrolytic metalloenzyme to incorporate iron, as opposed to zinc, 17,18 presumably because, unlike other metal ions, Zn 2+ is not complicated by redox chemistry. Iron, on the other hand, can promote unwanted side reactions with dioxygen (i.e., Fenton chemistry involving OH · radicals) upon reduction to the Fe 2+ oxidation state. 19,20 The NHase iron site is, however, redox inactive and stabilized in the 3+ oxidation state. The stabilization of Fe 3+ is accomplished by placing the iron in an electron-rich environment consisting of five anionic ligands-two deprotonated peptide amides and three cysteinates (Figure 1). Two of the three coordinated cysteinate sulfurs are oxidized (post-translationally modified) in NHase -one to a sulfenic acid ( 114 Cys-S-(OH)) and the other to a sulfinate 112 CysSO 2 −.3,21 The third cysteinate sulfur, which is trans to the inhibitor/substrate binding site and less accessible to solvent, remains unmodifie...

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“…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)(13)(14).…”

Section: Nitrile Hydratases (Nhases)mentioning

confidence: 99%

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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Cobalt(III) Complexes with Carboxamido-N and Sulfenato-S or Sulfinato-S Ligands Suggest that a Coordinated Sulfenate-S is Essential for the Catalytic Activity of Nitrile Hydratases

Abstract: A bis(sulfenato-S)Co III complex and its corresponding bis(sulfinato-S) complex were prepared. Their catalytic nitrile hydration activity was tested. Whereas the former cata-

Cited by 64 publications

References 28 publications

Post‐translational modification of Rhodococcus R312 and Comamonas NI1 nitrile hydratases

Post‐translational modification of Rhodococcus R312 and Comamonas NI1 nitrile hydratases

How Does Single Oxygen Atom Addition Affect the Properties of an Fe−Nitrile Hydratase Analogue? The Compensatory Role of the Unmodified Thiolate

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

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