Synthesis, endogenous detection, and mitochondrial function of the hydroxy-substituted Coenzyme Q10 derivative HO-Q10
2019
Hochschulschrift
Zugriff:
Quinones are redox-active molecules playing important roles in all organisms. In humans, the para-benzoquinone derivative Coenzyme Q10 (CoQ10) carrying a chain of 10 isoprene units and two neighbouring methoxy groups at the benzoquinone ring is found ubiquitously. It is an essential electron and proton transporter in the mitochondrial respiratory chain and fulfils various important functions like regulating redox homeostasis and membrane viscosity. In 2011, Bogeski et al. chemically modified the functional head group of CoQ10 exchanging one methoxy group by a hydroxy group. The hydroxy analogue can also be produced by CYP450 present in mitochondria and endoplasmic reticulum and is a postulated intermediate in CoQ biosynthesis that had not been detected in eukaryotes. The aim of this thesis was to gain first insights into the biological role of the mono-demethylated Coenzyme Q10. Therefore, within this study and my precedent master thesis, the CoQ10 derivative, HO-CoQ10, was synthesized and purified in sufficient amounts for the first time. Its structure could be verified using mass spectrometry and 1H/13C 2-dimensional nuclear magnetic resonance spectroscopy. The synthesis was confirmed to always produce a constitutional isomer mixture of HO-CoQ10 modified at the 2- or 3-position of the quinone ring. Due to the high lipophilicity mediated by the long isoprene chain, reliable transition to the aqueous phase was crucial for experiments. An ethanolic solution of 1 mM CoQ10 and 5 mM HO-CoQ10 was stable at room temperature and could be diluted in aqueous media. Hence, most experiments were conducted with 1% ethanol resulting in a maximal concentration of 10 μM CoQ10. To understand the physiological importance of HO-CoQ10, its endogenous occurrence was clarified using ultra-high-pressure liquid chromatography coupled to tandem mass spectrometry. In isopropanol extracts from crude bovine heart mitochondria, HO-CoQ10 was detected for the first time and estimated to have a concentration of 100 μM in mitochondrial membranes. Evaluating toxicity of exogenously applied HO-CoQ10, no effect on cancer cell lines cultivated under standard cell culture conditions was found: No interference with metabolic activity and proliferation was observed in HeLa, MelJuso and Jurkat T cells using the CellTiter-Blue® reduction assay and apoptosis was not induced in Jurkat T cells analysing caspase activity using Caper-GR sensor. Detecting HO-CoQ10 in mitochondria and considering the essential role of CoQ10 in the electron transport chain, its influence on respiration was evaluated measuring oxygen consumption of isolated cardiac mitochondria from BL6N mice using a Clark-type electrode. HO-CoQ10 inhibited Complex I-, II-, and III-linked respiration. Surprisingly, also the native substance CoQ10 intervened with Complex I- and II-linked respiration, but to a lower extent than HO-CoQ10. Extramitochondrial calcium enhanced inhibition via CoQ10 and HO-CoQ10 by the same factor. Since respiration buffers contained inorganic phosphate affecting free metal concentrations, free calcium was defined using the fluorescent calcium indicator fura-2. Photometric activity assays of respiratory chain enzymes from bovine heart mitochondria showed that HO-CoQ10 but not CoQ10 inhibited both oxidoreductase activity of Complex I and II. Decyl-ubiquinol:cytochrome c oxidoreductase (Complex III) was unaffected. In-gel activity staining of Complex I and II did not show interference on oxidase activities indicating that HO-CoQ10 acts on Q-binding sites of the complexes. Since CoQ10 is an important antioxidant in vivo but also shows prooxidant activity under distinct circumstances depending on its redox state, H2O2 production in the supernatant of coupled mouse heart mitochondria was examined using an HRP-based assay with Amplex® UltraRed. HO-CoQ10 increased Complex I- and II-linked ROS (reactive oxygen species) formation rate. CI-linked rates were similar for HO-CoQ10 and the ubiquinone- binding site inhibitor rotenone. In contrast, production rates induced by the Qi-site inhibitor of Complex III, antimycin A, were considerably higher suggesting a distinct mechanism of ROS formation. Glycerol dehydrogenase-linked H2O2 production was increased by antimycin A and slightly reduced by HO-CoQ10. To assess the correlation of respiration and ROS production, oxygen consumption and superoxide production were detected simultaneously by electron spin resonance spectroscopy with the oxygen sensor trityl and the spin probe for superoxide CMH. Similar to rotenone, CM• formation rate was not influenced by HO-CoQ10. Electron distribution in Complex I-associated iron-sulphur clusters of NADH-energized bovine heart mitochondria, visualised with low-temperature electron spin resonance spectroscopy, was not altered by HO- CoQ10. This work provides the first insight into the biological significance of the neglected hydroxy analogue of CoQ10. Focussing on mitochondrial respiration, the most studied and biologically important role of Coenzyme Q, it was found that both CoQ10 and HO-CoQ10 inhibit respiration. Enrichment of the substances was shown to decrease membrane fluidity in other studies, which might be the underlying mechanism diminishing electron transfer efficiency. Calcium potentiating the inhibition by both substances supports the observation that HO-CoQ10 as well as CoQ10 are able to bind calcium. Due to its lower redox potential, HO-CoQ10 has been predicted to be a more potent antioxidant with a potential to substitute CoQ10 in the medication of ROS related diseases. However, it was found to inhibit activity of respiratory chain complexes and stimulated ROS production most likely via blocking Q-binding sites. Downregulation of respiration and stimulation of ROS production by HO-CoQ10, found in small portions in metabolic active heart tissue, suggests a role in regulation of energy metabolism and might act as an internal brake for growth when synthesis is upregulated.
Titel: |
Synthesis, endogenous detection, and mitochondrial function of the hydroxy-substituted Coenzyme Q10 derivative HO-Q10
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Autor/in / Beteiligte Person: | Slowik, Ewa |
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Veröffentlichung: | 2019 |
Medientyp: | Hochschulschrift |
DOI: | 10.22028/D291-32037 |
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