Biochemical characterization of the inner membrane cytochrome CbcL: a gate for extracellular electron transfer in Geobacter sulfurreducens

• Autor(a) principal: Antunes, Jorge Manuel Andrade
• Data de Publicação: 2022
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/133794
• topic: Geobacter; extracellular electron transfer; inner membrane cytochromes; protein interactions; nuclear magnetic resonance; Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias

Exoelectrogenic microorganisms are in the spotlight due to their particular respiratory mechanisms and potential application in several biotechnological fields, including bioremediation, bioenergy production and microbial electrosynthesis. All these emergent applications rely on the capability of exoelectrogens to perform extracellular electron transfer, a mechanism that allows the bacteria to transfer electrons to the cell’s exterior. These respiratory pathways encompass different multiheme cytochromes along the inner membrane, periplasmic space, and outer membrane. CbcL from Geobacter sulfurreducens is an inner membrane associated multiheme cytochrome that plays an essential role in the transfer of electrons to final electron acceptors with a low redox potential, as Fe(III) oxides and electrodes poised at -100 mV. CbcL has a membrane di-heme b-type cytochrome domain with six transmembrane helices, linked to a periplasmic cytochrome domain with nine c-type heme groups. In this thesis, heterologous expression tests were performed for CbcL´s periplasmic domain. Its purification was also optimized by different types of chromatography and complementary spectroscopic techniques were used in its structural and functional characterization. Circular dichroism in the Far-UV region of the spectrum was used to determine the thermal stability of the cytochrome and predict its secondary structural elements. The protein was found to have a high percentage of disordered regions, as previously observed for other multiheme proteins. The reduction potentials of CbcL´s periplasmic domain were determined by potentiometric titrations followed by UV-visible spectroscopy at pH 7 and 8, and the obtained values allow a thermodynamically favorable transfer of electrons to its putative redox partners. Nuclear magnetic resonance was used to determine the spin state of the heme groups and to probe the biomolecular interactions between CbcL and the periplasmic triheme cytochrome PpcA. Crystallization tests were also carried out for future determination of CbcL´s periplasmic domain structure. The results obtained in this thesis show for the first time how electrons are injected into the periplasm of Geobacter sulfurreducens, allowing their subsequent transfer to the cell’s exterior.