Nitrile hydratases (NHases) are thiolate-ligated Fe(III)- or Co(III)containing enzymes, which convert nitriles to the corresponding amide under mild conditions. Proposed NHase mechanisms involve MOM-NCR, M(III)-OH, M(III)-iminol, and M(III)-amide intermediates. There have been no reported crystallographically characterized examples of these key intermediates. Spectroscopic and kinetic data support the involvement of a M(III)-NCR intermediate. A H-bonding network facilitates this enzymatic reaction. Herein we describe two biomimetic Co(III) NHase analogues that hydrate MeCN, and four crystallographically characterized NHase intermediate analogues, [Co [Co-III((SN4)-N-Me2(tren))(MeCN)](2+) (1), [Co-III((SN4)-N-Me2(tren))(OH)](+) (3), [Co-III((SN4)-N-Me2(tren))(NHC(O)CH3)](+) (2), and [Co-III((ON4)-N-Me2(tren))(NHC(OH)CH3)](2+) (5). Iminol-bound 5 represents the first example of a Co(III)-iminol compound in any ligand environment. Kinetic parameters (k(1)(298 K) = 2.98(5) M-1 s(-1), Delta H double dagger = 12.65(3) kcal/mol, Delta S double dagger = -14(7) e.u.) for nitrile hydration by I are reported, and the activation energy E-a = 13.2 kcal/mol is compared with that (E-a = 5.5 kcal/mol) of the NHase enzyme. A mechanism involving initial exchange of the bound MeCN for OH- is ruled out by the fact that nitrile exchange from 1 (k(ex)(300 K) = 7.3(1) x 10(-3) s(-1)) is 2 orders of magnitude slower than nitrile hydration, and that hydroxide bound 3 does not promote nitrile hydration. Reactivity of an analogue that incorporates an alkoxide as a mimic of the highly conserved NHase serine residue shows that this moiety facilitates nitrile hydration under milder conditions. Hydrogen-bonding to the alkoxide stabilizes a Co(III)-iminol intermediate. Comparison of the thiolate versus alkoxide intermediate structures shows that C N bond activation and C=O bond formation proceed further along the reaction coordinate when a thiolate is incorporated into the coordination sphere.