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...
Nitrile hydratases (NHase) are non-heme Fe(III)-containing, or noncorrinoid Co(III)-containing, microbial enzymes that catalyze nitrile hydration. 1 The iron form has been studied most extensively. The Fe(III) active site is low spin (S ) 1 / 2 ), and ligated by three cysteinates, two peptide amide nitrogens and either a hydroxide or an NO. [2][3][4] Given the high amount of sequence homology in the active site region, it is likely that the Co-NHase active site is virtually identical to Fe-NHase. 1,5 In one of two recent Fe NHase crystal structures 2,4 two of the metal-bound sulfurs appear to be oxidized, one to a sulfenate ( 114 S cys dO) and the other to a sulfinate ( 112 S cys (dO) 2 ). 4 The sulfenate is not observed by mass spectrometry. 6 Sulfenic acids are usually unstable, 7 and metal-sulfenates are readily oxidized to metalsulfinates. 8,9 A few synthetic NHase models containing oxidized sulfurs have been reported; 10-12 however, none of these incorporate a sulfenate, and only one 12 has an open coordination site.Our group has shown that the spin-state and spectroscopic properties of Fe-NHase can be nicely reproduced by six-coordinate Fe(III) model complexes containing two cis-thiolates and imines. 3,13-15 These models lack oxidized sulfurs, yet their spectroscopic properties are remarkably similar to the enzyme, suggesting that two, of the three, cysteinate NHase sulfurs remain unmodified. To understand how the sulfinate and, possibly, the sulfenate sulfur influences the electronic and reactivity properties of NHase, we have synthesized a series of sulfur-ligated, fivecoordinate Co(III) model complexes containing progressively more oxidized sulfurs.Five-coordinate [Co(III)(S 2 Me2 N 3 (Pr,Pr))] + (1) was synthesized in the same manner as its iron analogue. 15 Complex 1 is intermediate spin (S ) 1) over the temperature range 50-300 K (supplemental Figure S-1), and is reversibly reduced at E 1/2 ) -460 mV vs SCE. The average Co-S distance (2.16(2) Å) in 1 (Figure 1) 16 is shorter than most Co(III) thiolates (average ) 2.24 Å). [17][18][19] Azide and SCN -bind quantitatively to 1 at room temperature trans to one of the thiolate sulfurs. 20 Trigonal bipyramidal 1 (τ ) 0.87) 21 is converted to a more square pyramidal (τ ) 0.48) sulfinate/thiolate-ligated complex, [Co(III)(S Me2 (S O2 )N 3 (Pr,Pr))] + (2; Figure 2), 22 upon stirring in air for 3 days. Only one of the two thiolate sulfurs (S(2)) is oxidized, even upon prolonged stirring. Oxidation of S(2) causes the spinstate to change, from S ) 1 (in 1) to 0 (in 2), and the reduction potential to shift cathodically to E 1/2 ) -380 mV vs SCE. The mean S(2)-O(1,2) distance (1.453(2) Å) in 2 falls in the usual range (1.42-1.48 Å). 17,23,24 The Co-S(2) distance in 2 is indistinguishable from Co-S(1) (Figure 2). Both of the Co-S bonds in 2 are slightly shorter than the Co-S bonds in 1, because (1) Kobayashi, M.; Shimizu, S. Nature Biotechnol. 1998, 16, 733-36. (2) Huang, W.; Jia, J.; Cummings, J.; Nelson, M.; Schneider, G.; Lindqvist, Y. Structure 1997, 5, 691-6...
Detailed spectroscopic and computational studies of the low-spin iron complexes [Fe III (S 2 Me2 N 3 (Pr,Pr))(N 3 )] (1) and [Fe III (S 2 Me2 N 3 (Pr,Pr))] 1+ (2) were performed to investigate the unique electronic features of these species and their relation to the low-spin ferric active sites of nitrile hydratases. Low-temperature UV/vis/NIR and MCD spectra of 1 and 2 reflect electronic structures that are dominated by antibonding interactions of the Fe 3d manifold and the equatorial thiolate S 3p orbitals. The six-coordinate complex 1 exhibits a low-energy S π f Fe 3d xy (∼13000 cm -1 ) charge-transfer transition that results predominantly from the low energy of the singly occupied Fe 3d xy orbital, due to pure π interactions between this acceptor orbital and both thiolate donor ligands in the equatorial plane. The 3d π f 3d σ ligand-field transitions in this species occur at higher energies (>15000 cm -1 ), reflecting its nearoctahedral symmetry. The Fe 3d xz,yz f Fe 3d xy (d π f d π ) transition occurs in the near-IR and probes the Fe III −S π-donor bond; this transition reveals vibronic structure that reflects the strength of this bond (ν e ≈ 340 cm -1 ). In contrast, the ligand-field transitions of the five-coordinate complex 2 are generally at low energy, and the S π f Fe charge-transfer transitions occur at much higher energies relative to those in 1. This reflects changes in thiolate bonding in the equatorial plane involving the Fe 3d xy and Fe 3d x 2 -y 2 orbitals. The spectroscopic data lead to a simple bonding model that focuses on the σ and π interactions between the ferric ion and the equatorial thiolate ligands, which depend on the S−Fe−S bond angle in each of the complexes. These electronic descriptions provide insight into the unusual S ) 1 / 2 ground spin state of these complexes: the orientation of the thiolate ligands in these complexes restricts their π-donor interactions to the equatorial plane and enforces a low-spin state. These anisotropic orbital considerations provide some intriguing insights into the possible electronic interactions at the active site of nitrile hydratases and form the foundation for further studies into these low-spin ferric enzymes.
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