The Role of Mixed Amine/Amide Ligation in Nickel Superoxide Dismutase
- Univ. of Massachusetts, Amherst, MA (United States). Dept. of Chemistry
- Univ. of Wisconsin-Madison, Madison, WI (United States). Dept. of Chemistry
- Brookhaven National Lab. (BNL), Upton, NY (United States). Dept. of Chemistry
Superoxide dismutases (SODs) utilize a ping-pong mechanism in which a redoxactive metal cycles between oxidized and reduced forms that differ by one electron in order to catalyze the disproportionation of superoxide to dioxygen and hydrogen peroxide. Nickel dependent SOD (NiSOD) is a unique biological solution for controlling superoxide levels. This enzyme relies on the use of cysteinate ligands to bring the Ni(III/II) redox couple into the range required for catalysis (~300 mV). The use of cysteine thiolates, which are not found in any other SOD, is a curious choice because of their well-known oxidation by peroxide and dioxygen. The NiSOD active site cysteinate ligands are resistant to oxidation, and prior studies of synthetic and computational models point to the backbone N-donors in the active site (the N-terminal amine and the amide N atom of Cys2) as being involved in stabilizing the cysteines to oxidation. To test the role of the backbone N-donors, we have constructed a variant of NiSOD wherein an alanine residue is added to the N-terminus (Ala0-NiSOD), effectively altering the amine ligand to an amide. X-ray absorption, electronic absorption, and magnetic circular dichroism (MCD) spectroscopic analyses of as-isolated Ala0-NiSOD coupled with DFT geometry optimized models that were evaluated on the basis of the spectroscopic data within the framework of DFT and TD-DFT computations, are consistent with a diamagnetic Ni(II) site with two cysteinate, one His1 amide and one Cys2 amidate ligands. The variant protein is catalytically inactive, has an altered electronic absorption spectrum associated with the nickel site, and is sensitive to oxidation. Mass spectrometric analysis of the protein exposed to air shows the presence of a mixture of oxidation products, the principal ones being a disulfide, a bis-sulfenate, and a bis sulfinate derived from the active site cysteine ligands. Details of the electronic structure of the Ni(III) site available from the DFT calculations point to subtle changes in the unpaired spin density on the S-donors as being responsible for the altered sensitivity of Ala0-NiSOD to O2.
- Research Organization:
- Brookhaven National Laboratory (BNL), Upton, NY (United States); Univ. of Wisconsin - Madison, Madison, WI (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC), Basic Energy Sciences (BES). Chemical Sciences, Geosciences, and Biosciences Division; National Science Foundation (NSF); National Institutes of Health (NIH)
- Contributing Organization:
- University of Massachusetts - Amherst Mass Spectronomy Center
- Grant/Contract Number:
- SC0012704; CHE-1111462; GM 64631; T32-GM008505; CHE-0840494
- OSTI ID:
- 1476287
- Alternate ID(s):
- OSTI ID: 1571912
- Report Number(s):
- BNL-209119-2018-JAAM
- Journal Information:
- Inorganic Chemistry, Vol. 57, Issue 20; ISSN 0020-1669
- Publisher:
- American Chemical Society (ACS)Copyright Statement
- Country of Publication:
- United States
- Language:
- English
Web of Science
A Solvent‐Exposed Cysteine Forms a Peculiar Ni II ‐Binding Site in the Metallochaperone CooT from Rhodospirillum rubrum
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journal | October 2019 |
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