Shearer J, Kung IY, Lovell S, Kaminsky W, Kovacs JA. Why is there an "Inert" metal center in the active site of nitrile hydratase? Reactivity and ligand dissociation from a five-coordinate Co(III) nitrile hydratase model. J. Am. Chem. Soc. 123 (2001) 463-468.

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To determine how a substantially inert metal can play a catalytic role in the metalloenzyme nitrile hydratase (NHase), a reactive five-coordinate CoIII thiolate complex ([CoIII(S2Me2N3(Pr,Pr))](PF6) (1)) that resembles the active site of cobalt containing nitrile hydratase (Co NHase) was prepared. This was screened for reactivity, by using low-temperature electronic absorption spectroscopy, toward a number of biologically relevant "substrates". it was determined 1 will react with azide, thiocyanate, and ammonia, but is unreactive towards nitriles, NO, and butyrate. Substrate-bound 1 has similar spectroscopic and structural properties as [CoIII(ADIT2](PF6) (2). Complex 2 is a six-coordinate CoIII complex containing cis-thiolates and imine nitrogens, and has properties similar to the cobalt center of Co NHase. Substrate binding to 1 is reversible and temperature-dependent, allowing for the determination of the thermodynamic parameters of azide and thiocyanate binding and the rates of ligand dissociation. Azide and thiocyanate bind trans to thiolate, and with similar entropies and enthalpies (thiocyanate: dH = -7.5 +- 1.1 kcal/mol. dS = -17.2 +- 3.2 eu; azide: dH = -6.5 +- 1.0 kcal/mol, dS = -12.6 +- 2.4 eu). The rates of azide and thiozyanate displacement from the metal center are also comparable to one another (kd = (7.22 +- 0.04) x 10-1 s-1 for thiocyanate and kd = (2.14 +- 0.50) x 10-2 s-1 for azide), and are considerably faster than one would expect for a low-spin d6 six-coordinate CoIII complex. These rates are comparable to those of an analogous Fe(III) complex, demonstrating that Co(III) and Fe(III) react at comparable rates when in this ligand environment. This study therefore indicates that ligand displacement from a low-spin CoIII center in a ligand environment that resembles NHase is not prohibitively slow so as to disallow catalytic action in nonredox active cobalt metalloenzymes.


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