Journal
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
Volume 137, Issue 28, Pages 9044-9052Publisher
AMER CHEMICAL SOC
DOI: 10.1021/jacs.5b03629
Keywords
-
Categories
Funding
- National Science Foundation [CHE-0809188]
- Department of Energy, Office of Biological and Environmental Research
- National Institutes of Health
- US DOE Office of Science, Division of Chemical Sciences, Geosciences and Biosciences [DE-AC02-98CH10886]
- Direct For Mathematical & Physical Scien
- Division Of Chemistry [1111462] Funding Source: National Science Foundation
Ask authors/readers for more resources
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, N Delta 5-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 similar to 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 similar to 11% of the Ni in the sample and is similar to WT-NiSOD, but more axial, with g(z) < g(x,y). N-14-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 prediction based on synthetic and computational models.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available