NiII Complex Formation and Protonation States at the Active Site of a Nickel Superoxide Dismutase‐Derived Metallopeptide: Implications for the Mechanism of Superoxide Degradation. Issue 59 (21st September 2018)

• Record Type: Journal Article
• Authors: Tietze, Daniel; Koley Seth, Banabithi; Brauser, Matthias; Tietze, Alesia A.; Buntkowsky, Gerd
• Is Part Of: Chemistry. Volume 24:Issue 59(2018)
• Page Start: 15879
• Page End: 15888
• Publication Date: 2018-09-21
• Subjects: enzyme catalysis -- enzyme models -- metalloenzymes -- NMR spectroscopy -- reaction mechanisms; Chemistry -- Periodicals; 540
• DOI: 10.1002/chem.201803042
• Languages: English
• ISSNs: 0947-6539

Abstract

Abstract: A small, catalytically active metallopeptide (Nim 6 SOD, m 6 SOD=ACDLAC), which was derived from the nickel superoxide dismutase (NiSOD) active site was employed to study the mechanism of superoxide degradation, especially focusing on the protonation states of the Ni II donor atoms, the proton source, and the role of the N‐terminal proton(s). Therefore, the Ni II ‐metallopeptide was studied at various pHs and temperatures using UV/Vis and NMR spectroscopy. These studies indicate a strong reduction of the p K a of the Ni II ‐ligating donor atoms, resulting in a fully deprotonated Ni II active‐site environment. Furthermore, no titratable proton could be observed within a pH ranging from 6.5 to 10.5. This rules out a recently discussed adiabatic proton tunneling‐like hydrogen‐atom transfer process for the metallopeptides, not found in the native enzyme. Furthermore, variable‐temperature 1 H NMR measurements uncovered an extended hydrogen‐bond network within the Ni II active site of the metallopeptide similar to the enzyme. With respect to the deprotonated Ni II active site, the residual N‐terminal proton, which is a prerequisite for catalytic activity, cannot act as proton source. Most likely, it stabilizes the Ni II ‐coordinated substrate in an end‐on fashion, thus allowing for an inner‐sphere electron transfer. Lastly, and unlike the enzyme, the catalytic rate constant of superoxide degradation by the metallopeptides was determined to be strongly pH dependent, suggesting bulk water to be directly involved in proton donation, which in turn strongly suggests the N‐terminal histidine to be the respective proton donor in the enzyme. Abstract : Active‐site analysis : The Ni II active‐site protonation and mechanism of superoxide degradation was revealed by NMR spectroscopy and stopped‐flow kinetics employing a NiSOD‐derived metallopeptide (see figure). The results indicate that the active site is fully deprotonated upon Ni‐complex formation, resembling the hydrogen‐bond network of the enzyme but, unlike the enzyme, using water as proton source for superoxide reduction.