New insights into the mechanism of nickel superoxide degradation from studies of model peptides

A series of small, catalytically active metallopeptides, which were derived from the nickel superoxide dismutase (NiSOD) active site were employed to study the mechanism of superoxide degradation especially focusing on the role of the axial imidazole ligand. In the literature, there are contradicting propositions about the catalytic importance of the N-terminal histidine. Therefore, we studied the stability and activity of a set of eight NiSOD model peptides, which represent the major model systems discussed in the literature to date, yet differing in their length and their Ni-coordination. UV-Vis-coupled stopped-flow kinetic measurements and mass spectrometry analysis unveiled their high oxidation sensitivity in the presence of oxygen and superoxide resulting into a much faster Ni(II)-peptide degradation for the amine/amide Ni(II) coordination than for the catalytically inactive bis-amidate Ni(II) coordination. With respect to these results we determined the catalytic activities for all NiSOD mimics studied herein, which turned out to be in almost the same range of about 2 × 106 M−1 s−1. From these experiments, we concluded that the amine/amide Ni(II) coordination is clearly the key factor for catalytic activity. Finally, we were able to clarify the role of the N-terminal histidine and to resolve the contradictory literature propositions, reported in previous studies.


Analytical data for the NiSOD model peptides HPLC chromatograms and mass spectra of the linear peptides
UV-Vis spectra of NiSOD model peptides Figure S9: normalized UV-Vis spectra of the NiSOD mimics a) 1 (black trace), 7 (red trace) and 8 (light blue trace) and b) of 4 (blue trace), 3 (purple trace), 2 (orange trace), 5 (magenta trace) and 6 (green trace) in buffer (150 mM phosphate buffer, pH 8, 25°C). The inset shows the characteristic sulfur-to-Ni(II) ligand field transition at 460 nm.

Determination of the stability of the Ni-peptides
Peptide oxidation in air shown for Nim 9 SOD (a) (b) Figure S10: Oxidation process mediated by air exemplarily shown for Nim 9 SOD. a) overlay of HPLC chromatograms of Nim 9 SOD under various conditions. HPLC samples were prepared from 20 µL of the respective peptide solution in 80 µL water (0.1% TFA). black tracem 9 SOD in water pH 3 (not degassed), blue trace -Nim 9 SOD in buffer (250 mM, phosphate, pH 7.8). The chromatogram was recorded immediately after Ni addition. red tracesample from blue trace was aged for 24 hours on air. b) ESI mass spectra recorded from the peaks ranging from 8.799 to 9.420 min from the aged Nim 9 SOD sample (red trace  4+ ) m 9 SOD. Please note, that the acidic conditions of the HPLC eluent system remove the Ni-ion from the peptide, thus the Ni-peptide complex is not observable under these conditions.

Peptide treatment with KO2
Figure S19: LC-MS analysis of Nim 7 SOD (1 mM, phosphate buffer 150 mM, pH 8.0) treated with KO2. a) resulting HPLC chromatogram of the peptide after treatment with KO2 b) Mass spectra for the peptide peaks indicated (colored lines) in a). c) experimental and simulated isotope patterns of the peptide species, which were identified in the KO2/peptide solution. [M] corresponds to the mass of the linear peptide minus two protons.

Figure S20
: LC-MS analysis of Nim 7 SOD H1H' (1 mM, phosphate buffer 150 mM, pH 8.0) treated with KO2. a) resulting HPLC chromatogram of the peptide after treatment with KO2 b) Mass spectra for the peptide peaks indicated (colored lines) in a). c) experimental and simulated isotope patterns of the peptide species, which were identified in the KO2/peptide solution.
[M] corresponds to the mass of the linear peptide minus two protons.

Figure S21
: LC-MS analysis of Nim 7 SOD H1A (1 mM, phosphate buffer 150 mM, pH 8.0) treated with KO2. a) resulting HPLC chromatogram of the peptide after treatment with KO2 b) Mass spectra for the peptide peaks indicated (colored lines) in a). c) experimental and simulated isotope patterns of the peptide species, which were identified in the KO2/peptide solution.
[M] corresponds to the mass of the linear peptide minus two protons.

Figure S22
: LC-MS analysis of Nim 7 SOD AcHis (1 mM, phosphate buffer 150 mM, pH 8.0) treated with KO2. a) resulting HPLC chromatogram of the peptide after treatment with KO2 b) Mass spectra for the peptide peaks indicated (colored lines) in a). c) experimental and simulated isotope patterns of the peptide species, which were identified in the KO2/peptide solution.
[M] corresponds to the mass of the linear peptide minus two protons.

Figure S23
: LC-MS analysis of Nim 7 SOD H1H-tos (1 mM, phosphate buffer 150 mM, pH 8.0) treated with KO2. a) resulting HPLC chromatogram of the peptide after treatment with KO2 b) Mass spectra for the peptide peaks indicated (colored lines) in a). c) experimental and simulated isotope patterns of the peptide species, which were identified in the KO2/peptide solution.
[M] corresponds to the mass of the linear peptide minus two protons. Figure S24: LC-MS analysis of Nim 9 SOD (1 mM, phosphate buffer 150 mM, pH 8.0) treated with KO2. a) resulting HPLC chromatogram of the peptide after treatment with KO2 b) Mass spectra for the peptide peaks indicated (colored lines) in a). c) experimental and simulated isotope patterns of the peptide species, which were identified in the KO2/peptide solution.
[M] corresponds to the mass of the linear peptide minus two protons.

Figure S25
: LC-MS analysis of Nim 12 SOD (1 mM, phosphate buffer 150 mM, pH 8.0) treated with KO2. a) resulting HPLC chromatogram of the peptide after treatment with KO2 b) Mass spectra for the peptide peaks indicated (colored lines) in a). c) experimental and simulated isotope patterns of the peptide species, which were identified in the KO2/peptide solution.
[M] corresponds to the mass of the linear peptide minus two protons.       Figure S32: Color coded amplitude of flexibility at the NiSOD active site according to the crystallographic b-factor calculated from average root mean square fluctuations. Comparison of a) NiSOD H1Q and b) wt. NiSOD. c) superposition of the active site of wt. NiSOD (orange) and NiSOD H1Q (colored according to Figure 6) d) root mean square deviation (r.m.s.d.) of NiSOD with respect to the starting structure. e) structural mobility of the imidazole side chain of His1 with respect to the imidazole Nδ1 -Ni(II) distance as a function of simulation time.