Biochemical characterization of the inner membrane cytochrome CbcL: a gate for extracellular electron transfer in Geobacter sulfurreducens
Autor(a) principal: | |
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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 |
Resumo: | 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. |
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Biochemical characterization of the inner membrane cytochrome CbcL: a gate for extracellular electron transfer in Geobacter sulfurreducensGeobacterextracellular electron transferinner membrane cytochromesprotein interactionsnuclear magnetic resonanceDomínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e TecnologiasExoelectrogenic 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.Microrganismos exoeletrogénicos estão atualmente em destaque devido às suas invulgares cadeias respiratórias e a sua potencial aplicação em várias áreas da biotecnologia, incluindo biorremediação, produção de bioenergia e eletrossíntese microbiana. Todas estas aplicações exploram a capacidade que estas bactérias apresentam para transferir eletrões do espaço intracelular para o ambiente exterior, um processo designado por transferência extracelular de eletrões. Estas vias respiratórias englobam diferentes citocromos multi-hémicos localizados na membrana interna, espaço periplasmático e na membrana externa. O citocromo CbcL da bactéria Geobacter sulfurreducens é um citocromo multi-hémico associado à membrana interna que desempenha um papel essencial na transferência de eletrões para aceitadores finais com um baixo potencial redox, como óxidos de Ferro(III) e elétrodos posicionados a -100 mV. O CbcL possui dois domínios, um transmembranar e um solúvel. O primeiro é constituído por um citocromo di-hémico do tipo b e seis hélices transmembranares que o conectam ao domínio periplasmático que contém nove grupos hémicos do tipo c. Nesta tese foram efetuados testes de expressão heteróloga para o domínio periplasmático do CbcL. Foi também otimizada a sua purificação por diferentes tipos de cromatografia e utilizadas diversas técnicas espetroscópicas na sua caracterização estrutural e funcional. A técnica de dicroísmo circular foi usada para determinar a estabilidade térmica do citocromo e prever os seus elementos de estrutura secundária. Verificou-se que a proteína apresenta uma elevada percentagem de regiões desordenadas, tal como anteriormente observado para outras proteínas multi-hémicas. Os potenciais de redução do domínio periplasmático foram determinados por titulações potenciométricas seguidas por espectroscopia de UV-visível a pH 7 e 8, tendo-se verificado que o valores obtidos permitem a transferência termodinamicamente favorável de eletrões para os seus possíveis parceiros redox. A técnica de ressonância magnética nuclear foi usada para determinar o estado de spin dos hemos e investigar as interações biomoleculares com o citocromo periplasmático trihémico PpcA. Testes de cristalização foram também efetuados para futura determinação da sua estrutura. Os resultados obtidos nesta tese mostram pela primeira vez como os eletrões são injetados no periplasma de Geobacter sulfurreducens, permitindo a sua subsequente transferência para o exterior da célula.Morgado, Maria LeonorSalgueiro, CarlosRUNAntunes, Jorge Manuel Andrade2022-02-172025-02-01T00:00:00Z2022-02-17T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisapplication/pdfhttp://hdl.handle.net/10362/133794enginfo:eu-repo/semantics/embargoedAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-03-11T05:12:25Zoai:run.unl.pt:10362/133794Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T03:47:56.481876Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Biochemical characterization of the inner membrane cytochrome CbcL: a gate for extracellular electron transfer in Geobacter sulfurreducens |
title |
Biochemical characterization of the inner membrane cytochrome CbcL: a gate for extracellular electron transfer in Geobacter sulfurreducens |
spellingShingle |
Biochemical characterization of the inner membrane cytochrome CbcL: a gate for extracellular electron transfer in Geobacter sulfurreducens Antunes, Jorge Manuel Andrade Geobacter extracellular electron transfer inner membrane cytochromes protein interactions nuclear magnetic resonance Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias |
title_short |
Biochemical characterization of the inner membrane cytochrome CbcL: a gate for extracellular electron transfer in Geobacter sulfurreducens |
title_full |
Biochemical characterization of the inner membrane cytochrome CbcL: a gate for extracellular electron transfer in Geobacter sulfurreducens |
title_fullStr |
Biochemical characterization of the inner membrane cytochrome CbcL: a gate for extracellular electron transfer in Geobacter sulfurreducens |
title_full_unstemmed |
Biochemical characterization of the inner membrane cytochrome CbcL: a gate for extracellular electron transfer in Geobacter sulfurreducens |
title_sort |
Biochemical characterization of the inner membrane cytochrome CbcL: a gate for extracellular electron transfer in Geobacter sulfurreducens |
author |
Antunes, Jorge Manuel Andrade |
author_facet |
Antunes, Jorge Manuel Andrade |
author_role |
author |
dc.contributor.none.fl_str_mv |
Morgado, Maria Leonor Salgueiro, Carlos RUN |
dc.contributor.author.fl_str_mv |
Antunes, Jorge Manuel Andrade |
dc.subject.por.fl_str_mv |
Geobacter extracellular electron transfer inner membrane cytochromes protein interactions nuclear magnetic resonance Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias |
topic |
Geobacter extracellular electron transfer inner membrane cytochromes protein interactions nuclear magnetic resonance Domínio/Área Científica::Engenharia e Tecnologia::Outras Engenharias e Tecnologias |
description |
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. |
publishDate |
2022 |
dc.date.none.fl_str_mv |
2022-02-17 2022-02-17T00:00:00Z 2025-02-01T00:00:00Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
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publishedVersion |
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http://hdl.handle.net/10362/133794 |
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http://hdl.handle.net/10362/133794 |
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eng |
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eng |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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