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Title: 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 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 N 2S 2-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, withmore » g z < g x,y. 14N-hyperfine is observed on g z« less
 [1] ;  [2] ;  [3] ;  [1] ;  [4] ;  [1]
  1. Univ. of Massachusetts, Amherst, MA (United States)
  2. Lex Company Research Lab., Shirley, MA (United States)
  3. New England Peptide, Gardner, MA (United States)
  4. Brookhaven National Lab. (BNL), Upton, NY (United States)
Publication Date:
Report Number(s):
BNL-108507-2015-JA; BNL-111846-2016-JA
Journal ID: ISSN 0002-7863; R&D Project: CO004; KC0304030
Grant/Contract Number:
SC00112704; SC0012704; AC02-98CH10886
Accepted Manuscript
Journal Name:
Journal of the American Chemical Society
Additional Journal Information:
Journal Volume: 137; Journal Issue: 28; Journal ID: ISSN 0002-7863
American Chemical Society (ACS)
Research Org:
Brookhaven National Laboratory (BNL), Upton, NY (United States)
Sponsoring Org:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
Country of Publication:
United States
OSTI Identifier:
Alternate Identifier(s):
OSTI ID: 1335404