A Semisynthetic Strategy Leads to Alteration of the Backbone Amidate Ligand in the NiSOD Active Site

Computational investigations have implicated the amidate ligand in nickel superoxide dismutase (NiSOD) in stabilizing Ni-centered redox catalysis and in preventing cysteine thiolate ligand oxidation. To test these predictions, we have used an experimental approach utilizing a semisynthetic scheme th...

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Bibliographic Details
Published in:Journal of the American Chemical Society 2015-07, Vol.137 (28), p.9044-9052
Main Authors: Campeciño, Julius O, Dudycz, Lech W, Tumelty, David, Berg, Volker, Cabelli, Diane E, Maroney, Michael J
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Catalytic Domain
INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
Ligands
Models, Molecular
Mutagenesis
Nickel - chemistry
Nickel - metabolism
Oligopeptides - chemistry
Oligopeptides - metabolism
Oxidation-Reduction
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Sequence Deletion
Streptomyces - chemistry
Streptomyces - enzymology
Streptomyces - genetics
Streptomyces - metabolism
Superoxide Dismutase - chemistry
Superoxide Dismutase - genetics
Superoxide Dismutase - metabolism
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Summary:Computational investigations have implicated the amidate ligand in nickel superoxide dismutase (NiSOD) in stabilizing Ni-centered redox catalysis and in preventing cysteine thiolate ligand oxidation. To test these predictions, we have used an experimental approach utilizing a semisynthetic scheme that employs native chemical ligation of a pentapeptide (HCDLP) to recombinant S. coelicolor NiSOD lacking these N-terminal residues, NΔ5-NiSOD. Wild-type enzyme produced in this manner exhibits the characteristic spectral properties of recombinant WT-NiSOD and is as catalytically active. The semisynthetic scheme was also employed to construct a variant where the amidate ligand was converted to a secondary amine, H1*-NiSOD, a novel strategy that retains a backbone N-donor atom. The H1*-NiSOD variant was found to have only ∼1% of the catalytic activity of the recombinant wild-type enzyme, and had altered spectroscopic properties. X-ray absorption spectroscopy reveals a four-coordinate planar site with N2S2-donor ligands, consistent with electronic absorption spectroscopic results indicating that the Ni center in H1*-NiSOD is mostly reduced in the as-isolated sample, as opposed to 50:50 Ni­(II)/Ni­(III) mixture that is typical for the recombinant wild-type enzyme. The EPR spectrum of as-isolated H1*-NiSOD accounts for ∼11% of the Ni in the sample and is similar to WT-NiSOD, but more axial, with g z < g x,y . 14N-hyperfine is observed on g z , confirming the addition of the apical histidine ligand in the Ni­(III) complex. The altered electronic properties and implications for redox catalysis are discussed in light of predictions based on synthetic and computational models.
ISSN:0002-7863
1520-5126
DOI:10.1021/jacs.5b03629