Untersuchungen zum Mechanismus des katalytischen Superoxidabbaus durch die Nickel-Superoxiddismutase anhand funktioneller peptid-basierter Modellverbindungen
Zusammenfassung der Projektergebnisse
A series of NiSOD model peptides, which represent the major NiSOD models used to study the mechanism of superoxide degradation in the literature, were synthesized and their catalytic activities were assessed by stopped-flow kinetic experiments. Stopped-Flow kinetic measurements and LC-MS analysis were employed to assess the stability of the NiSOD model peptides against oxygen, hydrogen peroxide and superoxide. Intensive NMR and EPR analysis on the NiSOD model peptides were performed. The main results of this project can be summarized as follows: (1) Stopped-flow experiments revealed that the catalytic activities of the NiSOD model peptides are all on the same level, which unveiled that H1 is not required for the catalytic superoxide degradation by the NiSOD model peptides suggesting a different mechanism of superoxide degradation for the model peptides compared to the NiSOD enzyme. This solved a longstanding dispute in the scientific community. (2) Stopped-flow experiments and DFT calculations indicate a key role for the N-terminal amine protons with respect to the catalytically active mixed amine/amide Ni(II) coordination versus the inactive bis-amidate Ni(II) coordination. (3) The half-life of the NiSOD model peptides were quantified by stopped-flow kinetic measurements. Whereas the catalytically active peptides were reported to be rather short living the catalytically inactive bis-amidate NiSOD model peptide was about an order of magnitude more stable against oxidation. This can be explained by a more symmetric – thus more stable – coordination geometry of the later, which were obtained from DFT calculations. (4) NMR and EPR studies lead us to the conclusion that the line broadening of the NMR resonances of the NiSOD model peptides is supposed to be inhomogeneous and does not result from any paramagnetic Ni-species. Most likely, it results from the presence of multiple conformations/isomers in solution. (5) Solid-state NMR experiments indicated, that the NiSOD model peptides do not bind or contain any structurally important water molecules in contrast to our previous suggestion. (6) Solution NMR data uncovered the lack of any titratable proton within the Ni(II) active site of the model peptides over a pH range of 6.5 to 10.5. Accompanying stopped-flow experiments unveiled a strong pH dependence of the catalytic performance of the model peptides suggesting bulk water as the source of the proton for the superoxide reduction. (7) Finally, our studies strongly point towards the N-terminal His as source of the proton in the enzyme.
Projektbezogene Publikationen (Auswahl)
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(2018) NiII Complex Formation and Protonation States at the Active Site of a Nickel Superoxide Dismutase-Derived Metallopeptide: Implications for the Mechanism of Superoxide Degradation. Chemistry (Weinheim an der Bergstrasse, Germany) 24 (59) 15879–15888
Tietze, Daniel; Koley Seth, Banabithi; Brauser, Matthias; Tietze, Alesia A.; Buntkowsky, Gerd
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NMR Crystallography as a Novel Tool for the Understanding of the Mode of Action of Enzymes: SOD a Case Study. Appl. Magn. Reson. 2014, 45 (9), 841-857
Tietze, D.; Voigt, S.; Mollenhauer, D.; Buntkowsky, G.
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NMR Crystallography as a Novel Tool for the Understanding of the Mode of Action of Enzymes: SOD a Case Study. Appl. Magn. Reson. 2014, 45 (9), 841-857
Tietze, D.; Voigt, S.; Mollenhauer, D.; Buntkowsky, G.
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“New insights into the mechanism of nickel superoxide degradation from studies of model peptides”, Sci Rep 2017, 7, 17194
D. Tietze, J. Sartorius, B. Koley Seth, K. Herr, P. Heimer, D. Imhof, D. Mollenhauer, G. Buntkowsky,
