title: A Semisynthetic Strategy Leads to Alteration of the Backbone Amidate Ligand in the NiSOD Active Site.
abstract: 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 gz < gx,y. (14)N-hyperfine is observed on gz, 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.
authors.affiliation: †Department of Chemistry University of Massachusetts, Amherst, Massachusetts 01003, United States.
authors.forename: Julius O
authors.initials: JO
authors.lastname: Campeciño
authors.affiliation: ‡Lex Company Research Lab, Phoenix Park, 2 Shaker Road, Suite D 106, Shirley, Massachusetts 01464, United States.
authors.forename: Lech W
authors.initials: LW
authors.lastname: Dudycz
authors.affiliation: §New England Peptide, 65 Zub Lane, Gardner, Massachusetts 01440, United States.
authors.forename: David
authors.initials: D
authors.lastname: Tumelty
authors.affiliation: †Department of Chemistry University of Massachusetts, Amherst, Massachusetts 01003, United States.
authors.forename: Volker
authors.initials: V
authors.lastname: Berg
authors.affiliation: ∥Department of Chemistry, Brookhaven National Laboratory, Building 555A, P.O. Box 5000 Upton, New York 11973, United States.
authors.forename: Diane E
authors.initials: DE
authors.lastname: Cabelli
authors.affiliation: †Department of Chemistry University of Massachusetts, Amherst, Massachusetts 01003, United States.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Maroney
mesh_terms: D000595:Amino Acid Sequence; D020134:Catalytic Domain; D008024:Ligands; D008958:Models, Molecular; D016296:Mutagenesis; D009532:Nickel / Q000737:chemistry* / Q000378:metabolism; D009842:Oligopeptides / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013302:Streptomyces / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D008024:Ligands; D009842:Oligopeptides; D011994:Recombinant Proteins; D009532:Nickel; D013482:Superoxide Dismutase