title: Nitrile hydration by thiolate- and alkoxide-ligated Co-NHase analogues. Isolation of Co(III)-amidate and Co(III)-iminol intermediates.
abstract: 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 M(III)-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(III)(S(Me2)N(4)(tren))(MeCN)](2+) (1), [Co(III)(S(Me2)N(4)(tren))(OH)](+) (3), [Co(III)(S(Me2)N(4)(tren))(NHC(O)CH(3))](+) (2), and [Co(III)(O(Me2)N(4)(tren))(NHC(OH)CH(3))](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), ΔH(‡) = 12.65(3) kcal/mol, ΔS(‡) = -14(7) e.u.) for nitrile hydration by 1 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) × 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.
authors.affiliation: The Department of Chemistry, University of Washington, Box 351700, Seattle, Washington 98195-1700, United States.
authors.forename: Rodney D
authors.initials: RD
authors.lastname: Swartz
authors.forename: Michael K
authors.initials: MK
authors.lastname: Coggins
authors.forename: Werner
authors.initials: W
authors.lastname: Kaminsky
authors.forename: Julie A
authors.initials: JA
authors.lastname: Kovacs
mesh_terms: D003035:Cobalt / Q000737:chemistry*; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009570:Nitriles / Q000737:chemistry*; D013816:Thermodynamics; D014867:Water / Q000737:chemistry*
chemical_list: D009570:Nitriles; D014867:Water; D003035:Cobalt