Ligand binding in globins of Caenorhabditis elegans and Methanosarcina acetivorans : from over-expression to spectroscopic characterization
This thesis comprises studies on heme-containing proteins, including globin 33 (GLB-33) and GLB-3 from Caenorhabditis elegans (C. elegans), and the Protoglobin of Methanosarcina acetivorans (MaPgb), and their interactions with nitrite and nitric oxide (NO). Chapter 1 introduces globins, their structure, evolution, and roles in various organisms, emphasizing their interactions with reactive nitrogen species due to their physiological significance. The present state-of-the-art of the previously mentioned globins is reviewed, along with research objectives aligned with my supervisors' broader research lines. A brief theoretical overview of biochemical and biophysical methods used is included. Chapters 4 to 7 form the core of the thesis, presenting the main research results. Chapter 4 and 5 focus on the in-depth study of the globin domain of GLB-33 from C. elegans. Extensive examination of its interaction with nitrite is conducted using complementary spectroscopic techniques at varying pH levels. Additionally, a detailed investigation of the ferric hydroxide-ligated globin domain and ferrous NO-bound state is presented using electron paramagnetic resonance (EPR) spectroscopy. The results of both chapters are linked with a discussion on the role of amino acid Arg E10 in the process and its impact on the nitrite binding mode at the heme iron. Chapter 6 shifts the focus to the ferric MaPgb, highlighting its peculiar electronic state and NO-binding capacity. Kinetic analyses by UV-vis and spin-trap EPR spectroscopy reveal a high preference for NO over nitrite binding, and limited evidence of a potential nitrite dismutase activity of the globin. The final chapter of the results section focuses on my contribution to the first characterization study of the cysteine-rich GLB-3 from C. elegans. The experimental optimization to determine the redox potential of this unique globin is outlined, along with initial enzymatic tests, revealing limited reactivity towards small molecules and a strong bis-histidine coordination of GLB-3. Overall, this thesis contributes to our understanding of globins and their interactions with nitrite and NO from a spectroscopic perspective. The work highlights widely different behaviours and mechanisms among various globins, emphasizing the importance of the unique heme pocket structure on globin function and enhancing our understanding of these crucial proteins and their biochemical properties.
Antwerpen : Universiteit Antwerpen, Faculteit Wetenschappen, Departement Fysica , 2023
xix, 239 p.
Supervisor: Van Doorslaer, Sabine [Supervisor]
Supervisor: Dewilde, Sylvia [Supervisor]
Supervisor: Moens, Luc [Supervisor]
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The publisher created published version Available from 18.09.2024
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Creation 17.10.2023
Last edited 27.10.2023
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