title: Use of metallopeptide based mimics demonstrates that the metalloprotein nitrile hydratase requires two oxidized cysteinates for catalytic activity.
abstract: Nitrile hydratases (NHases) are non-heme Fe(III) or non-corrin Co(III) containing metalloenzymes that possess an N(2)S(3) ligand environment with nitrogen donors derived from amidates and sulfur donors derived from cysteinates. A closely related enzyme is thiocyanate hydrolase (SCNase), which possesses a nearly identical active-site coordination environment as CoNHase. These enzymes are redox inactive and perform hydrolytic reactions; SCNase hydrolyzes thiocyanate anions while NHase converts nitriles into amides. Herein an active CoNHase metallopeptide mimic, [Co(III)NHase-m1] (NHase-m1 = AcNH-CCDLP-CGVYD-PA-COOH), that contains Co(III) in a similar N(2)S(3) coordination environment as CoNHase is reported. [Co(III)NHase-m1] was characterized by electrospray ionization-mass spectrometry (ESI-MS), gel-permeation chromatography (GPC), Co K-edge X-ray absorption spectroscopy (Co-S: 2.21 Å; Co-N: 1.93 Å), vibrational, and optical spectroscopies. We find that [Co(III)NHase-m1] will perform the catalytic conversion of acrylonitrile into acrylamide with up to 58 turnovers observed after 18 h at 25 °C (pH 8.0). FTIR data used in concert with calculated vibrational data (mPWPW91/aug-cc-TZVPP) demonstrates that the active form of [Co(III)NHase-m1] has a ligated SO(2) (ν = 1091 cm(-1)) moiety and a ligated protonated SO(H) (ν = 928 cm(-1)) moiety; when only one oxygenated cysteinate ligand (i.e., a mono-SO(2) coordination motif) or the bis-SO(2) coordination motif are found within [Co(III)NHase-m1] no catalytic activity is observed. Calculations of the thermodynamics of ligand exchange (B3LYP/aug-cc-TZVPP) suggest that the reason for this is that the SO(2)/SO(H) equatorial ligand motif promotes both water dissociation from the Co(III)-center and nitrile coordination to the Co(III)-center. In contrast, the under- or overoxidized motifs will either strongly favor a five coordinate Co(III)-center or strongly favor water binding to the Co(III)-center over nitrile binding.
authors.affiliation: Department of Chemistry, University of Nevada, Reno, Nevada 89557, USA. shearer@unr.edu
authors.forename: Jason
authors.initials: J
authors.lastname: Shearer
authors.forename: Paige E
authors.initials: PE
authors.lastname: Callan
authors.forename: Justina
authors.initials: J
authors.lastname: Amie
mesh_terms: D055162:Biocatalysis; D003035:Cobalt / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry*; D006836:Hydro-Lyases / Q000737:chemistry / Q000378:metabolism*; D008968:Molecular Conformation; D018716:Molecular Mimicry; D009942:Organometallic Compounds / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry*; D011789:Quantum Theory; D013237:Stereoisomerism
chemical_list: D009942:Organometallic Compounds; D010455:Peptides; D003035:Cobalt; D006836:Hydro-Lyases; C048831:nitrile hydratase; D003545:Cysteine