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

Abstract

A disulfide-bridged peptide containing two Ni²⁺ binding sites based on the nickel superoxide dismutase protein, {Ni₂(SOD^mds)}, 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(SOD^mds)} reacts with O₂ at an ~3.5-fold faster rate than at pH = 7.4. Electronic structure calculations indicate 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.

Keywords: biological nickel sites; dinuclear nickel metallopeptides; nickel-thiolates; thiolate oxidative damage.

Figure 1.

Left: Analytical HPLC chromatogram of the crude mixture resulting from the synthesis of SOD^mds with a detector cutoff of 0 intensity units. Identifiable products are highlighted. A mobile phase of a mixture of 0.1% TFA in water and 0.1% TFA in acetonitrile and a linear gradient of 9:1 water:acetonitrile − 4:6 water:acetonitrile over the course of 30 min. Right: MALDI-TOF of the purified peptide SOD^mds (* indicates [SOD^mds]⁺ with the YDPA residues cleaved from the C-terminus of one of the monomers).

Figure 2.

Left: electronic absorption spectra (bottom) and CD spectra (top) of {Ni₂^II(SOD^mds)} at pH 7.4 and 9.6. The solid black spectra represent the experimental data, the red dashed curves represent the individual transitions deconvolved from the spectra and the dashed black spectra represent the convolution of the individual transitions. Right: pH profile showing the change in absorbance at 320 nm vs change in pH upon going from pH 7.4 to 9.6 (red circles) and pH 9.6 to 7.4 (blue squares).

Figure 3.

Left: XANES region of the Ni K-edge X-ray absorption spectrum for {Ni₂^II(SOD^mds)} at pH 7.4 (red) and 9.6 (blue). The inset depicts a blow-up of the Ni(1s → 3d) and Ni(1s → 4p_z) transitions. Right: Magnitude Fourier Transformed k³(χ) and unfiltered k³(χ) for {Ni₂^II(SOD^mds)} at pH 9.6 (A and B) and 7.4 (C and D). Refinements pH 7.4: a) Ni-S: n = 2; r = 2.1804(14) Å; σ² = 0.0026(2) Ų, b) Ni-N: n = 2; r = 1.907(16) Å; σ² = 0.0013(6) Ų; σ² = 0.0019(6) Ų; E_o = 8347.1 eV; ε² = 0.69. Refinements pH 9.6: a) Ni-S: n = 3; r = 2.229(2) Å; σ² = 0.0044(2) Ų, b) Ni-N: n = 1; r = 1.889(9) Å; σ² = 0.0013(8) Ų, c) Ni-Ni: n = 1; r = 3.25(3) Å; σ² = 0.0061(15) Ų; E_o = 8346.3 eV; ε² = 1.47.

Figure 4.

Sulfur K-edge XANES of {Ni₂^II(SOD^mds)} at high 7.4 (red) and 9.6 (blue).

Figure 5.

Computationally derived nickel site models of {Ni₂^II(SOD^mds)}. Metric parameters are provided next to the Ni-ligand bonds.

Figure 6.

Comparison of the TD-DFT calculated Ni K-edge XANES with the experimental spectra for {Ni₂(SOD^mds)} at pH 7.4 (left) and 9.6 (right).

Figure 7.

Left: Experimental (black spectrum) pH 7.4 and TD-DFT calculated S K-edge X-ray absorption spectra models of {Ni₂^II(SOD^mds)} (unprotonated model: gold spectrum; monoprotonated model: blue spectrum; doubly-protonated model: red spectrum). Right: Experimental (black spectrum) pH 9.6 and calculated spectrum (disulfide bridged model: red spectrum) of {Ni₂^II(SOD^mds)}.

Figure 8.

CD spectra following the air oxidation of {Ni₂^II(SOD^mds)} at pH 7.4 (left) and 9.6 (right), with the blue spectra representing the trace at t = 0 seconds, the red spectra representing the traces recorded every 600 seconds (10 min) over the course of 12 hours, and the teal spectra represent the CD spectra of the solutions following 24 hours of O₂ exposure. The insets depict the kinetics traces highlighting the decay of {Ni₂^II(SOD^mds)} (blue trace) and best fit of the kinetic trace to a first order rate law.

Figure 9.

A) Isosurface plots (0.03 a.u.) of the LUMO through HOMO-3 of the doubly-protonated (left) and unprotonated (right) computational models of the pH 7.4 form of the nickel-site of {Ni₂(SOD^mds)}. The energies were normalized to the non-bonding Ni(3d_z2) orbital, highlighted in red. B) Isosurface plots (0.03 a.u.) of the LUMO+1 through HOMO-5 of the disulfide bridged dinuclear {Ni₂(SOD^mds)} computational model.

Figure 10.

Structures of binuclear nickel-site models of {Ni₂^II(SOD^mds)} and {Ni₂^II(SOD^mds-S(H⁺)C1)}. The disulfide bridge and methylene groups have been represented as small spheres and wires for clarifty.

Chart 1.

Representative structures of the active sites of cysteinate-ligated nickel containing metalloproteins.

Scheme 1.

Interconversion of the structures of the nickel-sites of {Ni₂^II(SOD^mda)} at pH 7.4 and 9.6 and subsequent oxidative decomposition upon O₂ exposure.