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Metallopeptide based mimics with substituted histidines approximate a key hydrogen bonding network in the metalloenzyme nickel superoxide dismutase.

ABSTRACT: 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.

SUBMITTER: Shearer J

PROVIDER: S-EPMC2778858 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Metallopeptide based mimics with substituted histidines approximate a key hydrogen bonding network in the metalloenzyme nickel superoxide dismutase.

Shearer Jason J Neupane Kosh P KP Callan Paige E PE

Inorganic chemistry 20091101 22

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 el ...[more]

PMID: 19894770

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Project description:The active-site structures of the oxidized and reduced forms of manganese-substituted iron superoxide dismutase (Mn(Fe)SOD) are examined, for the first time, using a combination of spectroscopic and computational methods. On the basis of electronic absorption, circular dichroism (CD), magnetic CD (MCD), and variable-temperature variable-field MCD data obtained for oxidized Mn(Fe)SOD, we propose that the active site of this species is virtually identical to that of wild-type manganese SOD (MnSOD), with both containing a metal ion that resides in a trigonal bipyramidal ligand environment. This proposal is corroborated by quantum mechanical/molecular mechanical (QM/MM) computations performed on complete protein models of Mn(Fe)SOD in both its oxidized and reduced states and, for comparison, wild-type (WT) MnSOD. The major differences between the QM/MM optimized active sites of WT MnSOD and Mn(Fe)SOD are a smaller (His)N-Mn-N(His) equatorial angle and a longer (Gln146(69))NH···O(sol) H-bond distance in the metal-substituted protein. Importantly, these modest geometric differences are consistent with our spectroscopic data obtained for the oxidized proteins and high-field electron paramagnetic resonance spectra reported previously for reduced Mn(Fe)SOD and MnSOD. As Mn(Fe)SOD exhibits a reduction midpoint potential (E(m)) almost 700 mV higher than that of MnSOD, which has been shown to be sufficient for explaining the lack of SOD activity displayed by the metal-subtituted species (Vance, C. K.; Miller, A. F. Biochemistry 2001, 40, 13079-13087), E(m)'s were computed for our experimentally validated QM/MM optimized models of Mn(Fe)SOD and MnSOD. These computations properly reproduce the experimental trend and reveal that the drastically elevated E(m) of the metal substituted protein stems from a larger separation between the second-sphere Gln residue and the coordinated solvent in Mn(Fe)SOD relative to MnSOD, which causes a weakening of the corresponding H-bond interaction in the oxidized state and alleviates steric crowding in the reduced state.

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.

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Metallopeptide based mimics with substituted histidines approximate a key hydrogen bonding network in the metalloenzyme nickel superoxide dismutase.

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Metallopeptide based mimics with substituted histidines approximate a key hydrogen bonding network in the metalloenzyme nickel superoxide dismutase.

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