Dataset Information

Nickel superoxide dismutase: structural and functional roles of His1 and its H-bonding network.

ABSTRACT: Crystal structures of nickel-dependent superoxide dismutases (NiSODs) reveal the presence of a H-bonding network formed between the NH group of the apical imidazole ligand from His1 and the Glu17 carboxylate from a neighboring subunit in the hexameric enzyme. This interaction is supported by another intrasubunit H-bond between Glu17 and Arg47. In this study, four mutant NiSOD proteins were produced to experimentally evaluate the roles of this H-bonding network and compare the results with prior predictions from density functional theory calculations. The X-ray crystal structure of H1A-NiSOD, which lacks the apical ligand entirely, reveals that in the absence of the Glu17-His1 H-bond, the active site is disordered. Characterization of this variant using X-ray absorption spectroscopy (XAS) shows that Ni(II) is bound in the expected N2S2 planar coordination site. Despite these structural perturbations, the H1A-NiSOD variant retains 4% of wild-type (WT) NiSOD activity. Three other mutations were designed to preserve the apical imidazole ligand but perturb the H-bonding network: R47A-NiSOD, which lacks the intramolecular H-bonding interaction; E17R/R47A-NiSOD, which retains the intramolecular H-bond but lacks the intermolecular Glu17-His1 H-bond; and E17A/R47A-NiSOD, which lacks both H-bonding interactions. These variants were characterized by a combination of techniques, including XAS to probe the nickel site structure, kinetic studies employing pulse-radiolytic production of superoxide, and electron paramagnetic resonance to assess the Ni redox activity. The results indicate that in addition to the roles in redox tuning suggested on the basis of previous computational studies, the Glu17-His1 H-bond plays an important structural role in the proper folding of the "Ni-hook" motif that is a critical feature of the active site.

SUBMITTER: Ryan KC

PROVIDER: S-EPMC4405897 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Nickel superoxide dismutase: structural and functional roles of His1 and its H-bonding network.

Ryan Kelly C KC Guce Abigail I AI Johnson Olivia E OE Brunold Thomas C TC Cabelli Diane E DE Garman Scott C SC Maroney Michael J MJ

Biochemistry 20150121 4

Crystal structures of nickel-dependent superoxide dismutases (NiSODs) reveal the presence of a H-bonding network formed between the NH group of the apical imidazole ligand from His1 and the Glu17 carboxylate from a neighboring subunit in the hexameric enzyme. This interaction is supported by another intrasubunit H-bond between Glu17 and Arg47. In this study, four mutant NiSOD proteins were produced to experimentally evaluate the roles of this H-bonding network and compare the results with prior ...[more]

PMID: 25580509

Similar Datasets

Project description:Nickel superoxide dismutase (NiSOD) is a recently discovered superoxide dismutase that utilizes the Ni(III)/Ni(II) couple to facilitate the disproportionation of O(2)(*-) into H(2)O(2) and O(2). A key structural component of NiSOD is an elongated axial His-imidazole Ni(III) bond (2.3-2.6 A) that is the result of a H-bonding network between His(1), Glu(17), and Arg(47). Herein we utilize metallopeptide based mimics of NiSOD with His(1) epsilon-nitrogen substituted imidazoles to approximate the electronic influence of this H-bonding network ({Ni(III/II)(SOD(M1)-Im-X)} X = Me, H, DNP, and Tos; SOD(M1)-Im-X = H'CDLPCGVYDPA where H' is an N-substituted His). All reduced {Ni(II)(SOD(M1)-Im-X)} are similar to one another as assessed by electronic absorption spectroscopy, circular dichroism (CD) spectroscopy, and Ni K-edge x-ray absorption (XAS). This indicates that the change in His(1) is having little influence on the square-planar Ni(II)N(2)S(2) center. In contrast, changes to the axial His(1) ligand impart differential spectroscopic properties on the oxidized {Ni(III)(SOD(M1)-Im-X)} metallopeptides. Resonance Raman spectroscopy (405 nm excitation) in conjunction with a normal coordinate analysis indicates that as the axial His imidazole is made less Lewis basic there is an increase in Ni(III)-S bond strength in the equatorial plane, with force constants for the Ni-S bond trans to the amine ranging from 1.54 to 1.70 mdyn A(-1). The rhombic electron paramagnetic resonance (EPR) spectra of the four oxidized metallopeptides are all consistent with low-spin Ni(III) contained in a square pyramidal coordination environment, but show changes in the hyperfine coupling to (14)N along g(z). This is attributable to a reorientation of the g(z) vector in the more (along the Ni(III)-N(imidazole) bond) versus less (along the S-Ni(III)-N(amine) bond) Lewis basic imidazole bases. This reorientation of g(z) along the xy plane translates into a decrease in A(zz) by approximately 20 MHz. A decrease in Lewis-basicity of the axial imidazole also translates into a 2 orders of magnitude increase in SOD catalysis across the metallopeptide series, with k(cat) ranging from 6(1) x 10(6) M(-1) s(-1) for the metallopeptide with the most Lewis basic imidazole to 6(2) x 10(8) M(-1) s(-1) for the metallopeptide with the least basic imidazole. This likely results from a fine-tuning of the electron transfer properties of the Ni-center, which optimize it for SOD catalysis.

Project description:Superoxide dismutases (SODs) utilize a ping-pong mechanism in which a redox-active metal cycles between oxidized and reduced forms that differ by one electron 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 vs. NHE). 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 was 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 density functional theory (DFT) geometry optimized models that were evaluated on the basis of the spectroscopic data within the framework of DFT and time-dependent 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.

Dataset Information

Nickel superoxide dismutase: structural and functional roles of His1 and its H-bonding network.

Publications

Nickel superoxide dismutase: structural and functional roles of His1 and its H-bonding network.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets