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

To determine how a substitutionally inert metal can play a catalytic role i n the metalloenzyme nitrile hydratase (NHase), a reactive five-coordinate C o-III thiolate complex ([Co-III((S2N3)-N-Me2(Pr,Pr))](PF6) (1)) that resemb les the active site of cobalt containing nitrile hydratase (Co NHase) was p repared. This was screened for reactivity, by using low-temperature electro nic absorption spectroscopy, toward a number of biologically relevant "subs trates". It was determined 1 will react with azide, thiocyanate, and ammoni a, but is unreactive toward nitriles, NO, and butyrate. Substrate-bound 1 h as similar spectroscopic and structural properties as [Co-III(ADIT(2))](PF6 ) (2). Complex 2 is a six-coordinate Co-III complex containing cis-thiolate s and imine nitrogens, and has properties similar to the cobalt center of C o NHase. Substrate binding to 1 is reversible and temperature-dependent, al lowing for the determination of the thermodynamic parameters of azide and t hiocyanate binding and the rates of ligand dissociation. Azide and thiocyan ate bind trans to a thiolate, and with similar entropies and enthalpies (th iocyanate: DeltaH = -7.5 +/- 1.1 kcal/mol, DeltaS = -17.2 +/- 3.2 eu; azide : DeltaH = -6.5 +/- 1.0 kcal/mol, DeltaS = -12.6 +/- 2.4 eu). The rates of azide and thiocyanate displacement from the metal center are also comparabl e to one another (k(d) = (7.22 +/- 0.04) x 10(-1) s(-1) for thiocyanate and k(d) = (2.14 +/- 0.50) x 10(-2) s(-1) for azide), and are considerably fas ter than one would expect for a low-spin dh six-coordinate Co-III complex. These rates are comparable to those of an analogous Fe(III) complex, demons trating that Co(III) and Fe(III) react at comparable rates when in this lig and environment. This study therefore indicates that ligand displacement fr om a low-spin Co-III center in a ligand environment that resembles NHase is not prohibitivly slow so as to disallow catalytic action in nonredox activ e cobalt metalloenzymes.