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 FeIII or non-corrin CoIII containing metalloenzymes that possess an N2S3 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 CoN-Hase. 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, [CoIIINHase-m1] (NHase-m1 = AcNH-CCDLP-CGVYD-PA-COOH), that contains CoIII in a similar N2S3 coordination environment as is found in CoNHase/SCNase. [CoIIINHase-m1] was characterized by ESI-MS, 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 [CoIIINHase-m1] will perform the catalytic conversion of acrylonitrile into acrylamide with up to 58 turnovers observed after 18 hours 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 [CoIIINHase-m1] has a ligated SO2 (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-SO2 coordination motif) or the bis-SO2 coordination motif are found within [CoIIINHase-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 SO2/SO(H) equatorial ligand motif promotes both water dissociation from the CoIII-center and nitrile coordination to the CoIII-center. In contrast, the under- or overoxidized motifs will either strongly favor a five coordinate CoIII-center or strongly favor water binding to the CoIII-center over nitrile binding.

pH Dependent Reversible Formation of a Binuclear Ni2 Metal-Center within a Peptide Scaffold

“…This is similar to what has been reported for all NiSOD metallopeptides generated to date. For these NiSOD inspired peptides lacking the N-terminal thiolate group, cleavage of the apo-peptide from the resin and subsequent aerobic work-up and purification yield purified monomeric peptides free of disulfide bonds between the two cysteinate sulfur atoms corresponding to Cys2 and Cys6 in the wild-type NiSOD sequence [26,27,30,34]. Considering the only major modification between SOD mds and similar NiSOD inspired peptides generated to date is the presence of the 2-mercaptoacetate group, we suggest that the disulfide moiety within SOD mds results from the oxidative S-S bond formation between two N-terminal thiol groups from two different peptides.…”

“…In addition to mimicking the NiSOD active site, derivatives of the NiSOD inspired metallopeptides are capable of not only mimicking NiSOD, but also mimicking the active-site of cobalt containing NHase, and coordinating Cu 2+ [34,35]. This inspired us to further derivatize a NiSOD metallopeptide mimic, SOD m1 (SOD m1 = (SOD mds = HCDLP-CGVYDA-PA), in order to generate different metal-site structures.…”

Multiple States of Nitrile Hydratase from Rhodococcus equi TG328-2: Structural and Mechanistic Insights from Electron Paramagnetic Resonance and Density Functional Theory Studies

“…4,5 This geometry is conserved among all Fe-type and Co-type NHase enzymes. 1,6–8 The protonation states of the axial Cys and the post-translationally modified cysteine-sulfenic and cysteine-sulfenic acids were suggested to be Cys-S − , CysSOH, and a CysSO 2 − based on sulfur K-edge EXAFS and geometry-optimized density functional theory (DFT) calculations. 9 The post-translational oxidation of the two equatorial cysteine thiolate moieties to sulfenic and sulfenic acids is essential for catalytic activity.…”

Metal-centered oxidation decreases nitrile hydration activity of bioinspired (N2S3)Ru-PPh3 precatalysts

“…[1][2][3][4][5][6] The active site of NHase contains a mononuclear low-spin non-heme iron(III) or non-corrin cobalt(III) center in an unusual N 2 S 3 X type donor motif that contains two carboxamido nitrogens and three cysteine derived sulfur donors in distinct oxidation states; thiolate (RS -), sulfenate/sulfenic acid (RSO¯/RSOH) and sulfinate (RSO 2¯) as shown in Scheme 1. [7,8] Although asymmetric sulfur-oxidation is crucial for the catalytic activity of NHase, [9] the Co(III) peptide maquettes of Shearer [10] and our Ru(II) bioinspired catalysts [11,12] are the only two systems to have successfully implemented a mixed sulfenato/sulfinato donor set into a catalytically active synthetic system. In fact, the total number of functional NHase complexes remains small and limited to low-spin d 6 Co(III) complexes, with the exception of our low-spin d 6 Ru(II) catalysts.…”