Nikkeliont tartalmazó szuperoxid dizmutáz (SOD) enzimmodellek SOD aktivitásának vizsgálata = SOD activity studies of nickel containing superoxide dismutase (SOD) enzyme models

Bonczidai-Kelemen, Dóra and Fábián, István and Borsos, Attila and Lihi, Norbert (2025) Nikkeliont tartalmazó szuperoxid dizmutáz (SOD) enzimmodellek SOD aktivitásának vizsgálata = SOD activity studies of nickel containing superoxide dismutase (SOD) enzyme models. SCIENTIA ET SECURITAS. ISSN 3057-9759 (In Press)

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Abstract

A NiSOD (nikkel szuperoxid dizmutáz) enzim a legkésőbb izolált SOD enzim, amely a szuperoxid gyökanion bomlását katalizálja nagy hatékonysággal, ugyanakkor a dizmutálási reakció mechanizmusa még nem tisztázott. Ennek érdekében kutatómunkánk során a NiSOD enzim számos modellvegyületét vizsgáltuk. A peptidek nikkelkomplexeit különböző termodinamikai és szerkezetvizsgálati módszereknek vetettük alá. Tanulmányunk ezen komplexek SOD aktivitásának vizsgálatára irányul a McCord–Fridovich-módszer és stopped-flow technika segítségével. Megállapítottuk, hogy a dizmutálási ciklusban megjelenő Ni(III) komplex kialakulásának feltétele két tiolát-csoport együttes jelenléte, illetve, hogy a kis tagszámú modellek esetében a Ni(III) komplex intramolekuláris redoxidegradációja a SOD aktivitás elvesztését vonja magával. | Superoxide dismutase (SOD) enzymes catalyze the disproportionation reaction of the superoxide radical anion to hydrogen peroxide and molecular oxygen. The nickel-containing SOD (NiSOD) was isolated ca. 20 years ago, and its structure and coordination mode significantly differ from those of other SOD enzymes. Previous studies have shown that the first six amino acid residues (HCDLPC) of the N-terminal region are essential in metal binding, and its nickel complex exhibits the same coordination mode that is characteristic for the NiSOD enzyme. However, the mechanism of the dismutation remains uncovered. Therefore, we investigated the native NiSOD enzyme fragment (HCDLPCGVY, wtNiSOD) and its mutants. The peptides and their nickel complexes were studied by thermody- namic and several spectroscopic methods (pH-potentiometry, UV-Visible, CD, and EPR spectroscopy) and their SOD activities were assessed by the xanthine/xanthine-oxidase/NBT assay (McCord–Fridovich assay) and by dedicated stopped-flow measurements. While the McCord–Fridovich method is widely used, it provides only semi-quant- itative description of the SOD activity and does not provide information on the reaction mechanism. For this reason, we also examined the reaction between the Ni(II) complexes and the superoxide radical anion using stopped-flow techniques. In this study, we report the SOD activity measurements of six compounds modeling the NiSOD enzyme. By mod- ifying the wtNiSOD peptide chain, we obtained the following mutants: SODHH (HHDLPCGVY), SODHC (HCDLPHGVY), wtPen (HPenDLPCGVY), wtCC (CCDLPCGVY), and DIM (HCDLPCGVY-K-YVGCPLDCH), which is capable of binding two metal ions. Our results shown that the formation of the catalytically relevant Ni(III) complex requires the simultaneous existence of two cysteine residues. In accordance with this, the SODHH and SODHC mutants showed significantly lower SOD activity. The kinetic parameters obtained from the fitting of the kinetic traces indicated the appearance of a catalytically inactive component (Ni*). In the case of wtNiSOD, the complete conversion of the nickel complex to the Ni* form occurred within 50 ms, whereas for the wtPen, wtCC, and DIM mutants this transformation occurred in less than 20 ms. In NiSOD enzymes, intramolecular redox degra- dation does not need to be considered, so we assume that this process may be hindered due to secondary interactions originating from the structure of the enzyme.

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