Sulfur K-Edge XAS and DFT Calculations on Nitrile Hydratase:  Geometric and Electronic Structure of the Non-heme Iron Active Site

Dey, Abhishek; Chow, Marina S.; Taniguchi, Kayoko; Lugo‐Mas, Priscilla; Davin, Steven D.; Maeda, Mizuo; Kovacs, Julie A.; Odaka, Masafumi; Hodgson, Keith O.; Hedman, Britt; Solomon, Edward I.

doi:10.1021/ja0549695

Cited by 90 publications

(159 citation statements)

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“…Based on sulfur Kedge EXAFS and geometry-optimized DFT calculations, the protonation state of the sulfenic acid was suggested to be Cys-SOH. 33 Therefore, the loss of this hydrogen bond between αR157 and the O1-atom of the sulfenic acid ligand will likely decrease the nucleophilicity of the sulfenic oxygen atom, consistent with the observed blue-shift in the S → Fe(III) LMCT band. Since the sulfenic acid ligand has been shown to be capable of functioning as the nucleophile in the catalytic reaction, 12 the loss of the hydrogen bond between the αR157 and the O1-atom of the sulfenic acid ligand would be expected to significantly decrease the activity.…”

Section: Journal Of Biological Inorganicsupporting

confidence: 57%

“…This αCys 104 -SOH residue has been proposed to function as the nucleophile in catalysis, 12 and has been suggested to exist in its protonated form at pH 7.5. 33 Mutation of αR157 to lysine (αR157K), a residue that in principle could provide a hydrogen bond to the active site sulfenic acid ligand, resulted in an enzyme with a kcat value of 32 ± 4 s −1 and a Km value of 240 ± 10. These data suggest that the protonation state and stability of the active site sulfenic acid ligand is maintained through hydrogen-bond formation with αR157 and disruption of this hydrogen-bond likely result in deprotonation of the active site sulfenic acid ligand decreasing the ability of the catalytic active site to function.…”

Section: Resultsmentioning

confidence: 99%

“…The lack of this hydrogen bond likely results in a decrease in negative charge on the sulfenic acid oxygen as this oxygen has been suggested to be protonated at pH 7.5. 33 This would result in an increase in electron donation to the Fe(III) center from the sulfenic acid sulfur ligand. As a consequence of this change in π-electron donation from the sulfenic acid sulfur ligand, the amount of π-electron donation from the axial thiolate ligand to the Fe(III) ion would likely decrease.…”

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Analyzing the catalytic role of active site residues in the Fe-type nitrile hydratase from Comamonas testosteroni Ni1

Martinez

Krzywda

et al. 2015

J Biol Inorg Chem

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A strictly conserved active site arginine residue (αR157) and two histidine residues (αH80 and αH81) located near the active site of the Fe-type nitrile hydratase from Comamonas testosteroni Ni1 (CtNHase), were mutated. These mutant enzymes were examined for their ability to bind iron and hydrate acrylonitrile. For the αR157A mutant, the residual activity (kcat = 10 ± 2 s −1 ) accounts for less than 1 % of the wild-type activity (kcat = 1100 ± 30 s −1 ) while the Km value is nearly unchanged at 205 ± 10 mM. On the other hand, mutation of the active site pocket αH80 and αH81 residues to alanine resulted in enzymes with kcat values of 220 ± 40 and 77 ± 13 s −1 , respectively, and Km values of 187 ± 11 and 179 ± 18 mM. The double mutant (αH80A/αH81A) was also prepared and provided an enzyme with a kcat value of 132 ± 3 s −1 and a Km value of 213 ± 61 mM. These data indicate that all three residues are catalytically important, but not essential. X-ray crystal structures of the αH80A/αH81A, αH80W/αH81W, and αR157A mutant CtNHase enzymes were solved to 2.0, 2.8, and 2.5 Å resolutions, respectively. In each mutant enzyme, hydrogen-bonding interactions crucial for the catalytic function of the αCys 104 -SOH ligand are disrupted. Disruption of these hydrogen bonding interactions likely alters the nucleophilicity of the sulfenic acid oxygen and the Lewis acidity of the active site Fe(III) ion.

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Section: Journal Of Biological Inorganicsupporting

confidence: 57%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

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Analyzing the catalytic role of active site residues in the Fe-type nitrile hydratase from Comamonas testosteroni Ni1

Martinez

Krzywda

et al. 2015

J Biol Inorg Chem

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“…25 The sulfinate (RSO 2 − ) has been shown to remain unprotonated. 25 The enzyme becomes inactive when a second oxygen atom is added to the sulfenate 114 Cys-S-(OH), implying that the singly oxygenated sulfur plays a specific role in catalysis. 26 For this reason, the studies herein focus on the effect of single oxygen atom addition on properties likely to influence catalytic activity.…”

mentioning

confidence: 99%

“…However, sulfur K-edge X-ray absorption spectroscopy (XAS) studies suggest that the sulfenic acid residue is protonated. 25 The sulfinate (RSO 2 − ) has been shown to remain unprotonated. 25 The enzyme becomes inactive when a second oxygen atom is added to the sulfenate 114 Cys-S-(OH), implying that the singly oxygenated sulfur plays a specific role in catalysis.…”

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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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3‐Mercaptopropionate Dioxygenase (MDO)

Schmittou

Solano

Kiser

et al. 2023

Encyclopedia of Inorganic and Bioinorganic Chemistry

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Bacterial 3‐mercaptopropionate dioxygenase (MDO) is a mononuclear nonheme iron dioxygenase that catalyzes the O 2 ‐dependent oxidation of 3‐mercaptopropioniate (3MPA) to produce 3‐sulfinopropionate (3SPA). MDO is a member of the cupin superfamily of proteins and therefore shares a similar active site architecture as mammalian and bacterial cysteine dioxygenases (CDOs). However, unlike CDOs, which exhibit near exclusive specificity for l ‐cysteine (CYS), MDOs are more flexible with respect to accommodating other thiol‐bearing substrates of comparable size to 3MPA. The active site of MDO and CDO share two major structural features. First, they both contain a mononuclear nonheme iron site coordinated by three histidine residues. Since all protein‐derived ligands occupy one face of an octahedron, binding of dioxygen and organic substrate occurs at the opposite face of the 3‐His facial triad. Second, both enzymes share a conserved sequence of outer Fe‐coordination sphere amino acids (Ser, His, and Tyr) positioned adjacent to the iron site (∼ 4 Å). These residues participate in an extended proton relay network which ultimately donates an H‐bond to the enzymatic Fe‐site. To date, MDOs have only been identified among Gram‐negative soil proteobacteria. It is believed that the high concentration of 3MPA present in coastal sediments can be attributed to bacterial catabolism of organosulfur compounds present in saline environments. Indirect evidence also suggests that 3MPA may play a more central role in bacterial sulfur metabolism than currently understood.

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Sulfur K-Edge XAS and DFT Calculations on Nitrile Hydratase: Geometric and Electronic Structure of the Non-heme Iron Active Site

Cited by 90 publications

References 54 publications

Analyzing the catalytic role of active site residues in the Fe-type nitrile hydratase from Comamonas testosteroni Ni1

Analyzing the catalytic role of active site residues in the Fe-type nitrile hydratase from Comamonas testosteroni Ni1

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

3‐Mercaptopropionate Dioxygenase (MDO)

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