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Synthetic Models of Nitrile Hydratase

Nitrile Hydratase (NHase)1,2

Reaction

RCN + H2O --> RC(O)NH2

NHase catalyzes the hydrolysis of nitriles (RCN) to amides (RC(O)NH2). The enzyme is found in certain bacteria, such as Rhodococcus rhodochrous, and fungi that are capable of metabolizing nitriles as a sole source of nitrogen and carbon.

Relevance

NHase is used a biocatalyst for the industrial production of acrylamide from acrylonitrile. It has also been investigated for use in bioremediation. 

Stick drawing of NHase active siteActive Site

The active site of NHase contains either Fe(III) or Co(III). The metal ion is coordinated by two carboxamido nitrogens from the protein backbone and three cysteine derived sulfurs. Two of the sulfur donors are oxygenated through a post-translational modification yielding one sulfenato (RSO-) and one sulfinato (RSO2-) donor. The sixth coordination site is the presumed substrate binding site. It may be occupied by nitric oxide in an inactivated form of the enzyme.

Mechanism

The mechanism of this hydrolytic enzyme is still under debate. It is unclear which of the substrates (if either) coordinates directly to the metal ion during catalysis. It has been shown that sulfur-oxygenation is required for catalytic activity, although it is unclear what role this plays.

1. Kovacs, J. A. Chem. Rev. 2004, 104, 825-848. 2. Mascharak, P. K. Coord. Chem. Rev. 2002, 225, 201-214.

Research Efforts

Structural and Electronic Models

(bmmp-TASN)FeX

Our research centers on small molecule mimics of the NHase active. Complexes with the ligand (bmmp-TASN)2- reproduce key structural and electronic features of the enzyme. The pentadentate ligand allows for variation of a sixth ligand and numerous derivatives have been prepared. In this system, the spin-state of the complex is dependent on the nature of the sixth ligand and both high-spin and low-spin complexes are accessible.  

 

Sulfur Oxygenation3-5

RuSO2SOxrayThe observation of sulfur-oxygenates at the active site of NHase was counter to prior observations that air exposure of iron-thiolates typically leads to disulfide and rust. Through our model studies, we have found that the products of air exposure are dependent on the spin-state of the iron complex. For high-spin complexes, results are consistent with earlier expecations and iron-oxo clusters were isolated. For low-spin complexes, we find sulfur-oxygenation similar to that of NHase. Our computational investigations reveal that changes in the Fe-S bond covalency are responsible for these divergent pathways. Consistent with this explanation, the corresponding low-spin ruthenium derivative yields a family of sulfur-oxygenates upon oxygen exposure. The reaction products include the mixed sulfenato/sulfinato derivative, which mimics the active site of nitrile hydratase. 

Substrate Binding6

(LFe)2OH

Our model complexes are unique in their ability to bind either substrate (nitriles or water) for nitrile hydrolysis as well as the product (amides). Through a series of competition studies, we have found a preference for water > amides > nitriles. These results support a water-bound mechanism for NHase.

Functional Models

Our water-bound complex [(bmmp-TASN)FeOH2]+ is unreactive with nitriles. Deprotonation of the coordinated water results in isolation of the hydoxo- and oxo- complexes [[(bmmp-TASN)Fe]2OH]+ and [(bmmp-TASN)Fe]2O as a function of pH. Future efforts are aimed at developing functional models. 

 3. J. Biol. Inorg. Chem. 2008, 13, 1219-1230.  4. Inorg. Chem.  2010, 49, 5344-5346. 5. Inorg. Chem.  2010, 49, 10875-10881. 6. Inorg. Chem. 2009, 48, 2300-2308.


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