Cysteinate Protonation and Water Hydrogen Bonding at the Active-Site of a Nickel Superoxide Dismutase Metallopeptide-Based Mimic: Implications for the Mechanism of Superoxide Reduction

Shearer, Jason; Peck, Kristy L.; Schmitt, Jennifer C.; Neupane, Kosh P.

doi:10.1021/ja5079514

Cited by 24 publications

(43 citation statements)

References 51 publications

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“…As stated above, NiSOD itself has been shown to possess at least one Ni-S(H + )-Cys moiety in its reduced form [15]. Although not without controversy [33], we have provided strong evidence based on sulfur K-edge X-ray absorption studies that like NiSOD itself, NiSOD metallopeptide based mimics possess a Ni-S(H + )-Cys moiety at physiological pH as well [18,22]. Studies have also suggested that the pKa of the Ni-S(H + )-Cys proton within these active-sites is ~8.5, and can become reversibly deprotonated at high pH (>9.0) [22].…”

Section: Introductionsupporting

confidence: 62%

“…To date, two nickel containing metalloenzymes, nickel iron hydrogenase [NiFe]H 2 ase and nickel containing superoxide dismutase (NiSOD), have been demonstrated to possess at least one cysteinate ligand that becomes protonated, forming a Ni-S(H + )-Cys moiety under physiological conditions [15,16]. Concerning the role of the Ni-S(H + )-Cys moiety in biochemical reactions, it has been proposed that these moieties can behave as proton donors/acceptors and sources of formal hydrogen atoms [17][18][19][20][21][22]. However, their exact role(s) in biological reactions is currently unknown.…”

Section: Introductionmentioning

confidence: 99%

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pH Dependent Reversible Formation of a Binuclear Ni2 Metal-Center within a Peptide Scaffold

2019

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A disulfide-bridged peptide containing two Ni2+ binding sites based on the nickel superoxide dismutase protein, {Ni2(SODmds)} has been prepared. At physiological pH (7.4), it was found that the metal sites are mononuclear with a square planar NOS2 coordination environment with the two sulfur-based ligands derived from cysteinate residues, the nitrogen ligand derived from the amide backbone, and a water ligand. Furthermore, S K-edge X-ray absorption spectroscopy indicated that the two cysteinate sulfur atoms ligated to nickel are each protonated. Elevation of the pH to 9.6 results in the deprotonation of the cysteinate sulfur atoms, and yields a binuclear, cysteinate bridged Ni22+ center with each nickel contained in a distorted square planar geometry. At both pH = 7.4 and 9.6, the nickel sites are moderately air sensitive, yielding intractable oxidation products. However, at pH = 9.6, {Ni2(SODmds)} reacts with O2 at an ~3.5-fold faster rate than at pH = 7.4. Electronic structure calculations indicate that the reduced reactivity at pH = 7.4 is a result of a reduction in S(3p) character and deactivation of the nucleophilic frontier molecular orbitals upon cysteinate sulfur protonation.

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Section: Introductionsupporting

confidence: 62%

Section: Introductionmentioning

confidence: 99%

pH Dependent Reversible Formation of a Binuclear Ni2 Metal-Center within a Peptide Scaffold

2019

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“…The evidence comes primarily from sulfur K-edge X-ray absorption spectroscopy of as isolated enzyme as well as peptide mimics. 56,66,67 The sulfur K-edge data for the as isolated enzyme revealed two pre-edge features, 2469.7 eV and 2470.9 eV, (Figure 1.9) 23,67 ; and were assigned to S 1s →Ni(d z 2 ) and S 1s →Ni(d x 2 y 2 ), respectively. 23 The lower energy transition gradually disappeared upon continuous X-ray beam exposure of the sample as the Ni site is progressively photoreduced.…”

Section: ) That Leads Directly Towards Thementioning

confidence: 99%

“…The Cys6 methylated mimic also showed a sulfur K-edge spectrum reminiscent that of the Ni(II) M1 -H at pH 7.4. 66 This suggested that Cys6 is the probable protonation site.…”

Section: ) That Leads Directly Towards Thementioning

confidence: 99%

A Semisynthetic Strategy Leads to Alteration of the Backbone Amidate Ligand in the NiSOD Active Site

Campeciño

Dudycz

Tumelty³

et al. 2015

J. Am. Chem. Soc.

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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 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 ∼11% of the Ni in the sample and is similar to WT-NiSOD, but more axial, with gz < gx,y. (14)N-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 predictions based on synthetic and computational models.

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“…Questions concerning the enzyme's unusual N/S coordination sphere, particularly its stability toward O 2 and reactive oxygen species (ROS), in light of the reactive Ni–S(Cys) bonds, 13 and the catalytic mechanism have prompted researchers to develop maquette-based, 14–20 tripeptide, 21–24 and low molecular weight 25–38 analogues that seek to replicate NiSOD's structure and/or function. 39,40 Indeed, several experimental and theoretical efforts have suggested that the mixed peptide-N/amine-N ligation serves as one key factor to promote greater Ni-character in the redox-active molecular orbital of NiSOD, in particular for the Ni(II) or NiSOD red state, thus preventing unwanted S-oxidation/oxygenation.…”

Section: Introductionmentioning

confidence: 99%

Accessing Ni(III)-Thiolate Versus Ni(II)-Thiyl Bonding in a Family of Ni–N₂S₂ Synthetic Models of NiSOD

et al. 2015

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Superoxide dismutase (SOD) catalyzes the disproportionation of superoxide (O2• −) into H2O2 and O2(g) by toggling through different oxidation states of a first-row transition metal ion at its active site. Ni-containing SODs (NiSODs) are a distinct class of this family of metalloenzymes due to the unusual coordination sphere that is comprised of mixed N/S-ligands from peptide-N and cysteine-S donor atoms. A central goal of our research is to understand the factors that govern reactive oxygen species (ROS) stability of the Ni–S(Cys) bond in NiSOD utilizing a synthetic model approach. In light of the reactivity of metal-coordinated thiolates to ROS, several hypotheses have been proffered and include the coordination of His1-Nδ to the Ni(II) and Ni(III) forms of NiSOD, as well as hydrogen bonding or full protonation of a coordinated S(Cys). In this work, we present NiSOD analogues of the general formula [Ni(N2S)(SR′)]−, providing a variable location (SR′ = aryl thiolate) in the N2S2 basal plane coordination sphere where we have introduced o-amino and/or electron-withdrawing groups to intercept an oxidized Ni species. The synthesis, structure, and properties of the NiSOD model complexes (Et4N)[Ni(nmp)(SPh-o-NH2)] (2), (Et4N)[Ni(nmp)(SPh-o-NH2-p-CF3)] (3), (Et4N)[Ni(nmp)(SPh-p-NH2)] (4), and (Et4N)[Ni(nmp)(SPh-p-CF3)] (5) (nmp2− = dianion of N-(2-mercaptoethyl)picolinamide) are reported. NiSOD model complexes with amino groups positioned ortho to the aryl-S in SR′ (2 and 3) afford oxidized species (2ox and 3ox) that are best described as a resonance hybrid between Ni(III)-SR and Ni(II)-•SR based on ultraviolet–visible (UV-vis), magnetic circular dichroism (MCD), and electron paramagnetic resonance (EPR) spectroscopies, as well as density functional theory (DFT) calculations. The results presented here, demonstrating the high percentage of S(3p) character in the highest occupied molecular orbital (HOMO) of the four-coordinate reduced form of NiSOD (NiSODred), suggest that the transition from NiSODred to the five-coordinate oxidized form of NiSOD (NiSODox) may go through a four-coordinate Ni-•S(Cys) (NiSODox-Hisoff) that is stabilized by coordination to Ni(II).

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Cysteinate Protonation and Water Hydrogen Bonding at the Active-Site of a Nickel Superoxide Dismutase Metallopeptide-Based Mimic: Implications for the Mechanism of Superoxide Reduction

Cited by 24 publications

References 51 publications

pH Dependent Reversible Formation of a Binuclear Ni2 Metal-Center within a Peptide Scaffold

pH Dependent Reversible Formation of a Binuclear Ni2 Metal-Center within a Peptide Scaffold

A Semisynthetic Strategy Leads to Alteration of the Backbone Amidate Ligand in the NiSOD Active Site

Accessing Ni(III)-Thiolate Versus Ni(II)-Thiyl Bonding in a Family of Ni–N₂S₂ Synthetic Models of NiSOD

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Cysteinate Protonation and Water Hydrogen Bonding at the Active-Site of a Nickel Superoxide Dismutase Metallopeptide-Based Mimic: Implications for the Mechanism of Superoxide Reduction

Cited by 24 publications

References 51 publications

pH Dependent Reversible Formation of a Binuclear Ni2 Metal-Center within a Peptide Scaffold

pH Dependent Reversible Formation of a Binuclear Ni2 Metal-Center within a Peptide Scaffold

A Semisynthetic Strategy Leads to Alteration of the Backbone Amidate Ligand in the NiSOD Active Site

Accessing Ni(III)-Thiolate Versus Ni(II)-Thiyl Bonding in a Family of Ni–N2S2 Synthetic Models of NiSOD

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Accessing Ni(III)-Thiolate Versus Ni(II)-Thiyl Bonding in a Family of Ni–N₂S₂ Synthetic Models of NiSOD