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.

Figure 1

A) Reaction catalyzed by nitrile hydratases and B) reaction catalyzed by thiocyanate hydrolase. Note the similarity in purported active-site structures.

Figure 2

Electronic absorption spectrum of Co^II ligated NHase-m1. The inset depicts an expansion of the higher-energy ligand-field transitions.

Figure 3

FTIR spectra of [Co^IIINHase-m1] matured in the presence of ¹⁶O₂ (red) vs.¹⁸O₂ (blue) gas.

Figure 4

Top: XANES region of the X-ray absorption spectrum of [Co^IIINHase-m1] following 3 hours of maturation. Inset depicts the FF k³ data (Fourier Transformed from 2.2 – 14.2 k; back-transformed from 0.8 to 2.5 Å). Bottom: FT k³ data. In both the FT and FF k³ data the real data is represented as the solid red lines, the simulated data is depicted as the dashed blue line, and the difference spectra are depicted as the dashed green line. Best fits to the data: 3 N/O scatterers (1.93 Å σ² = 0.0062(8)Ų), 2 S scatterers (2.21 Å σ² = 0.0037(3)Ų); ε² = 0.82.

Figure 5

CD (top) and electronic absorption spectra (bottom) of [Co^IIINHase-m1] obtained in 10 mM NEM buffer at a pH = 8.0. The black solid spectrum represents the experimental data, the dashed red peaks represent the best-fit of the absorption and CD spectra decovolved into Gaussian line shapes, and the dashed blue spectrum represents the best-fit of the absorption and CD spectrum resulting from the sum of the Gaussian line shapes.

Figure 6

Infrared spectra of [Co^IIINHase-m1] recorded after 10 minutes (blue), 1 hour (green), 3 hours (red), and 5 hours (purple) of exposure to air.

Figure 7

Energy level diagram of the Co(3d) orbitals depicting the occupied (blue) and unoccupied (red) orbitals. Iso-surface plots of the corresponding molecular orbitals were generated with the molecular graphics program Molkel.

Figure 8

Simulated IR spectrum of 4 with ¹⁶O (red) and ¹⁸O (blue) labeling of the sulfinate and sulfenate. The sticks represent the individual transitions and the spectra was generated by applying Gaussian line shapes (50 cm⁻¹ peak width) to the transitions and summing them together.

Figure 9

Simulated CD spectra for 2-H₂O (A), 3-H₂O (B), 4-H₂O (C), and 4 (D). Simulated spectra are sums of Gaussian lineshapes applied to the individual transitions (red sticks) using a 1500 cm⁻¹ peak width.

Figure 10

Calculated free energies of ligand exchange normalized to the aqua species X-H₂O. The energetics of models 2 (black), 3 (blue), 4 (red), and 5 (green) are depicted.

Figure 11

Mayer bond orders (mPWPW91/aug-cc-TZVPP) calculated for the aqua-species X-H₂O (X = 2, 3, 4, and 5).

Scheme 1

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Scheme 4

Chart 1

Chart 1

Chart 2

Chart 2. Computational models examined in this study: A) (Co-NHase-SO₂-L)^2- (2-L), B) (Co-NHase-SO₂/SO-L)^2- (3-L), C) (Co-NHase-SO₂/SOH-L)^1- (4-L), D) (Co-NHase-2SO₂-L)^2- (5-L). In all cases L = H₂O (top), vacant (middle, abbreviated with no ligand description), or MeCN (bottom).