The geometric and electronic structure of the active site of the non-heme iron enzyme nitrile hydratase (NHase) is studied using sulfur K-edge XAS and DFT calculations. Using thiolate (RS − )-, sulfenate (RSO − )-, and sulfinate (RSO 2 − )-ligated model complexes to provide benchmark spectral parameters, the results show that the S K-edge XAS is sensitive to the oxidation state of S-containing ligands and that the spectrum of the RSO − species changes upon protonation as the S-O bond is elongated (by ~0.1 Å). These signature features are used to identify the three cysteine residues coordinated to the low-spin Fe III in the active site of NHase as CysS − , CysSOH, and CysSO 2 − both in the NO-bound inactive form and in the photolyzed active form. These results are correlated to geometry-optimized DFT calculations. The pre-edge region of the X-ray absorption spectrum is sensitive to the Z eff of the Fe and reveals that the Fe in [FeNO] 6 NHase species has a Z eff very similar to that of its photolyzed Fe III counterpart. DFT calculations reveal that this results from the strong π back-bonding into the π* antibonding orbital of NO, which shifts significant charge from the formally t 2 6 low-spin metal to the coordinated NO.

Nitrile hydratases (NHases) have an unusual iron or cobalt catalytic center with two oxidized cysteine ligands, cysteinesulfinic acid and cysteine-sulfenic acid, catalyzing the hydration of nitriles to amides. Recently, we found that the NHase of Rhodococcus erythropolis N771 exhibited an additional catalytic activity, converting tert-butylisonitrile (tBuNC) to tert-butylamine. Taking advantage of the slow reactivity of tBuNC and the photoreactivity of nitrosylated NHase, we present the first structural evidence for the catalytic mechanism of NHase with time-resolved x-ray crystallography. By monitoring the reaction with attenuated total reflectanceFourier transform infrared spectroscopy, the product from the isonitrile carbon was identified as a CO molecule. Crystals of nitrosylated inactive NHase were soaked with tBuNC. The catalytic reaction was initiated by photo-induced denitrosylation and stopped by flash cooling. tBuNC was first trapped at the hydrophobic pocket above the iron center and then coordinated to the iron ion at 120 min. At 440 min, the electron density of tBuNC was significantly altered, and a new electron density was observed near the isonitrile carbon as well as the sulfenate oxygen of ␣Cys 114. These results demonstrate that the substrate was coordinated to the iron and then attacked by a solvent molecule activated by ␣Cys -SOH.Nitrile hydratases (NHases) 2 catalyze the hydration of nitriles to the corresponding amides and are used as catalysts in the production of acrylamide, making them one of the most important industrial enzymes (1, 2). NHases contain a nonheme Fe 3ϩ or non-corrin Co 3ϩ catalytic center. Iron-type NHases show unique photoreactivity; the enzyme is inactivated by nitrosylation in the dark and immediately reactivated by photo-induced denitrosylation (3-5). The protein structure is highly conserved among all known NHases (6 -9) as well as a related enzyme, thiocyanate hydrolase (10). The metal site is also conserved, with a distorted octahedral geometry. All ligand residues are involved in a strictly conserved motif of the ␣ subunit, Cys 1 -Xaa-Leu-Cys 2 -Ser-Cys 3 , where two amide nitrogens of Ser and Cys 3 and three Cys sulfurs are coordinated to the metal (6). Cys 2 and Cys 3 are post-translationally modified to cysteine-sulfinic acid and cysteine-sulfenic acid, respectively (7), which probably take deprotonated forms at the metal site (11). The sixth ligand site is occupied by a solvent molecule (8) or by a NO molecule in nitrosylated iron-type NHase (7).Several reaction mechanisms have been proposed based on the protein structures (1, 6). First, nitriles directly bind to the metal to facilitate the nucleophilic attack of a water molecule on the nitrile carbon. In the other mechanisms, a water molecule activated by the metal directly or indirectly attacks nitriles trapped near the metal. In all cases, the metal is suspected to function as a Lewis acid. By reconstituting iron-type NHase from recombinant unmodified subunits, we demonstrated that the post-translational ...

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