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 in the metalloenzyme nitrile hydratase (NHase), a reactive five-coordinate Co(III) thiolate complex ([Co(III)(S(2)(Me2)N(3)(Pr,Pr))](PF(6)) (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 toward nitriles, NO, and butyrate. Substrate-bound 1 has similar spectroscopic and structural properties as [Co(III)(ADIT(2))](PF(6)) (2). Complex 2 is a six-coordinate Co(III) 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 a thiolate, and with similar entropies and enthalpies (thiocyanate: 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 comparable 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 faster than one would expect for a low-spin d(6) six-coordinate Co(III) 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 Co(III) center in a ligand environment that resembles NHase is not prohibitively slow so as to disallow catalytic action in nonredox active cobalt metalloenzymes.
Shearer
J
J
Contribution from the Department of Chemistry, University of Washington, Seattle, Washington 98195, USA.
Kung
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Lovell
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Kaminsky
W
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Kovacs
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JA
D001386:Azides / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D020134:Catalytic Domain; D003035:Cobalt / Q000737:chemistry; D018360:Crystallography, X-Ray; D006836:Hydro-Lyases / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D012988:Soil Microbiology; D013816:Thermodynamics; D013861:Thiocyanates / Q000378:metabolism
D001386:Azides; D001426:Bacterial Proteins; D008024:Ligands; D013861:Thiocyanates; D003035:Cobalt; D006836:Hydro-Lyases; C048831:nitrile hydratase