Nitrile hydratase. The first non-heme iron enzyme with a typical low-spin iron(III)-active center

Sugiura, Yukio; Kuwahara, Jun; Nagasawa, Tôru; Yamada, Hideaki

doi:10.1021/ja00253a046

Cited by 174 publications

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“…A similar charge-transfer band is also observed in the active form of Fe-NHase. 5,6 Under these conditions (methylene chloride, −90 °C), 1 does not appear to bind any nitriles or alcohols, and the only amine it will bind under these conditions is pyridine. With anionic ligands such as azide or thiocyanate, on the other hand, only 1 equiv of ligand is required to observe complete binding to 1 (and 2) at −90 °C in CH 2 Cl 2 .…”

Section: Resultsmentioning

confidence: 97%

“…The exchange rate (k ex ) was then determined by: (5) 4-tert-Butylpyridine was used to determine pyridine exchange rates from 1 and 2. In this case there is negligible unpaired spin density on the tert-butyl group carbons so that the exchange rate can be determined by: (6) The parameters ΔH ‡ and ΔS ‡ and E a were then determined through standard procedures. 20 …”

Section: Nmr Exchange Kineticsmentioning

confidence: 99%

“…13,41 There is spectroscopic evidence that supports mechanism 3. 6 Both the EPR signal and electronic absorption spectrum change upon the introduction of nitriles to NHase. 6 One could interpret this to mean that nitriles coordinate to the metal ion; however, it is also possible that the nitrile indirectly perturbs the metal ion by slightly distorting its structure when it "wedges" itself in the active-site pocket.…”

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The First Example of a Nitrile Hydratase Model Complex that Reversibly Binds Nitriles

et al. 2002

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Nitrile hydratase (NHase) is an iron-containing metalloenzyme that converts nitriles to amides. The mechanism by which this biochemical reaction occurs is unknown. One mechanism that has been proposed involves nucleophilic attack of an Fe-bound nitrile by water (or hydroxide). Reported herein is a five-coordinate model compound ([Fe III (S 2 Me2 N 3 (Et,Pr))] + ) containing Fe(III) in an environment resembling that of NHase, which reversibly binds a variety of nitriles, alcohols, amines, and thiocyanate. XAS shows that five-coordinate [Fe III (S 2 Me2 N 3 (Et,Pr))] + reacts with both methanol and acetonitrile to afford a six-coordinate solvent-bound complex.Competitive binding studies demonstrate that MeCN preferentially binds over ROH, suggesting that nitriles would be capable of displacing the H 2 O coordinated to the iron site of NHase. Thermodynamic parameters were determined for acetonitrile (ΔH = −6.2(±0.2) kcal/mol, ΔS = −29.4(±0.8) eu), benzonitrile (−4.2(±0.6) kcal/mol, ΔS = −18(±3) eu), and pyridine (ΔH = −8(±1) kcal/mol, ΔS = −41(±6) eu) binding to [Fe III (S 2 Me2 N 3 (Et,Pr))] + using variable-temperature electronic absorption spectroscopy. Ligand exchange kinetics were examined for acetonitrile, isopropylnitrile, benzonitrile, and 4-tert-butylpyridine using 13 C NMR line-broadening analysis, at a variety of temperatures. Activation parameters for ligand exchange were determined to be ΔH ‡ = 7.1(±0.8) kcal/mol, ΔS ‡ = −10(±1) eu (acetonitrile), ΔH ‡ = 5.4(±0.6) kcal/mol, ΔS ‡ = −17(±2) eu (iso-propionitrile), ΔH ‡ = 4.9(±0.8) kcal/mol, ΔS ‡ = −20(±3) eu (benzonitrile), and ΔH ‡ = 4.7(±1.4) kcal/mol ΔS ‡ = −18(±2) eu (4-tert-butylpyridine). The thermodynamic parameters for pyridine binding to a related complex, [Fe III (S 2 Me2 N 3 (Pr,Pr))] + (ΔH = −5.9(±0.8) kcal/mol, ΔS = −24(±3) eu), are also reported, as well as kinetic parameters for 4-tert-butylpyridine exchange (ΔH ‡ = 3.1(±0.8) kcal/mol, ΔS ‡ )−25(±3) eu). These data show for the first time that, when it is contained in a ligand environment similar to that of NHase, Fe(III) is capable of forming a stable complex with nitriles. Also, the rates of ligand exchange demonstrate that low-spin Fe(III) in this ligand environment is more labile than expected. Furthermore, comparison of (Tables S-6-S-9), van't Hoff plots for ligand binding to 1 and 2, variable-temperature electronic absorption spectra (Figures S-3-S-7), and EPR spectra for "substrate"-bound 2 , and crystallographic data for 2-NCS (Tables S-10-S-14) (PDF). This material is available free of charge via the Internet at http://pubs.acs.org. HHS Public AccessAuthor manuscript (S 2 Me2 N 3 (Pr,Pr))] + demonstrates how minor distortions induced by ligand constraints can dramatically alter the reactivity of a metal complex.Performing reactions under environmentally friendly conditions has recently become a desirable goal in the search for new catalysts. 1 Enzymes are often viewed as ideal in this respect because of their ability to perform chemical transformations und...

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Section: Resultsmentioning

confidence: 97%

Section: Nmr Exchange Kineticsmentioning

confidence: 99%

mentioning

confidence: 99%

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The First Example of a Nitrile Hydratase Model Complex that Reversibly Binds Nitriles

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“…However, none of these 35 metals was detected within the limits of the assay (20 ng/ml). In this respect, the isonitrile hydratase is quite different from the nitrile hydratases of Pseudomonas (27,28) and Brevibacterium (29) species, which contain tightly bound iron (1,30), and the nitrile hydratase of Rhodococcus rhodochrous J1, which contains cobalt as a prosthetic group (1,11,31).…”

Section: Resultsmentioning

confidence: 99%

Discovery of a Novel Enzyme, Isonitrile Hydratase, Involved in Nitrogen-Carbon Triple Bond Cleavage

Goda

Hashimoto

Shimizu

et al. 2001

Journal of Biological Chemistry

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Isonitrile containing an NϵC triple bond was degraded by microorganism sp. N19-2, which was isolated from soil through a 2-month acclimatization culture in the presence of this compound. The isonitrile-degrading microorganism was identified as Pseudomonas putida. The microbial degradation was found to proceed through an enzymatic reaction, the isonitrile being hydrated to the corresponding N-substituted formamide. The enzyme, named isonitrile hydratase, was purified and characterized. The native enzyme had a molecular mass of about 59 kDa and consisted of two identical subunits. The enzyme stoichiometrically catalyzed the hydration of cyclohexyl isocyanide (an isonitrile) to N-cyclohexylformamide, but no formation of other compounds was detected. The apparent K m value for cyclohexyl isocyanide was 16.2 mM. Although the enzyme acted on various isonitriles, no nitriles or amides were accepted as substrates.

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“…N-771. [38][39][40] These observed shifts have been attributed to a change in the π-back-donation from the axial cysteine ligand to the metal center, in response to perturbations at the active site. 41,42 Fig.…”

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confidence: 99%

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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Nitrile hydratase. The first non-heme iron enzyme with a typical low-spin iron(III)-active center

Cited by 174 publications

References 0 publications

The First Example of a Nitrile Hydratase Model Complex that Reversibly Binds Nitriles

The First Example of a Nitrile Hydratase Model Complex that Reversibly Binds Nitriles

Discovery of a Novel Enzyme, Isonitrile Hydratase, Involved in Nitrogen-Carbon Triple Bond Cleavage

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

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