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Biochemical and Physiological Insights Into Bacterial Cytochrome C Peroxidases From Cláudia Raquel da Silva Nóbrega Licenciatura em Biologia Molecular e Genética e Mestrado em Microbiologia Aplicada Biochemical and physiological insights into bacterial cytochrome c peroxidases from Escherichia coli and Neisseria gonorrhoeae Dissertação para obtenção do Grau de Doutor em Bioquímica, especialidade em Bioquímica Física Orientador: Doutora Sofia Rocha Pauleta, Investigadora Principal da Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa Co-orientador: Doutor Bart Devreese, Professor da Universidade de Ghent, Bélgica Júri: Presidente: Doutora Maria João Lobo dos Reis Madeira Crispim Romão Arguentes: Doutor Arsénio do Carmo Sales Mendes Fialho Doutor Carlos Alberto Gomes Salgueiro Vogais: Doutor João Paulo dos Santos Gomes Doutora Maria João Lobo dos Reis Madeira Crispim Romão Doutora Sofia Rocha Pauleta Setembro 2017 2017 Biochemical and physiological insights into bacterial cytochrome c peroxidases from Escherichia coli and Neisseria gonorrhoeae Cláudia Nóbrega Cláudia Raquel da Silva Nóbrega Licenciatura em Biologia Molecular e Genética e Mestrado em Microbiologia Aplicada Biochemical and physiological insights into bacterial cytochrome c peroxidases from Escherichia coli and Neisseria gonorrhoeae Dissertação para obtenção do Grau de Doutor em Bioquímica, especialidade em Bioquímica Física Orientador: Doutora Sofia Rocha Pauleta, Investigadora Principal da Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa Co-orientador: Doutor Bart Devreese, Professor da Universidade de Ghent, Bélgica. Júri: Presidente: Doutora Maria João Lobo dos Reis Madeira Crispim Romão Arguentes: Doutor Arsénio do Carmo Sales Mendes Fialho Doutor Carlos Alberto Gomes Salgueiro Vogais: Doutor João Paulo dos Santos Gomes Doutora Maria João Lobo dos Reis Madeira Crispim Romão Doutora Sofia Rocha Pauleta Setembro 2017 Biochemical and physiological insights into bacterial cytochrome c peroxidases from Escherichia coli and Neisseria gonorrhoeae Copyright by Cláudia Raquel da Silva Nóbrega, Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa e Universidade Nova de Lisboa Setembro 2017 A Faculdade de Ciências e Tecnologia e a Universidade Nova de Lisboa tem o direito, perpétuo e sem limites geográficos, de arquivar e publicar esta dissertação através de exemplares impressos reproduzidos em papel ou de forma digital, ou por qualquer outro meio conhecido ou que venha a ser inventado, e de a divulgar através de repositórios cientificos e de admitir a sua cópia e distribuição com objectivos educacionais ou de investigação, não comerciais, desde que seja dado crédito ao autor e editor. Acknowledgments I would like to express my sincere gratitude to all the people that made this thesis possible. All of them provided a valuable contribution to this journey and I can just hope that I may meet them again, someday. First of all, I would like to thank my supervisors. To my advisor Doctor Sofia Pauleta I have to thank for the opportunity to work in this project, for all that she taught me, her support, the time spent and much more, throughout all these years. I thank my co-advisor Prof. Bart Devreese for all the help with mass spectrometry analysis and for the uplifting and positive emails he has sent when I needed anything, always kind and available. I would like to show my appreciation to Prof. Isabel Moura and Prof. José Moura for allowing me to work in their labs and for sharing their knowledge and books. I thank to my collaborators from the X-Tal group, the crystallography group from UCIBIO, REQUIMTE. To Prof. Maria João Romão, who kindly received me in her lab and for providing new insightful ideas. To Doctor Ana Luísa Carvalho and Lina Juknaite for teaching me and helping me in all the crystallization experiments, but above all for never giving up and encouraging me to keep trying. I would also like to express my gratitude to the rest of the group, which was always available to help me and guide me. I thank to all my colleagues, the present and the past ones, for their advice, guidance, knowledge, help, inspiring discussions, friendship and innumerous special memories. I would like to express my gratitude especially to Cíntia Carreira, for being my PhD partner and a trusted friend. Words cannot describe how fortunate I am to have met her and how much she inspired me to be a better scientist. I thank Bernardo, my partner in life, for always, always, always, supporting me during my PhD thesis and for not complaining for all the long hours I made him wait before going home or for making him “wake up too early” to go to the lab. I am grateful to my brother, who inspired me to pursue a PhD and has always been available whenever I needed him. Agradeço aos meus pais que sempre me apoiaram, me motivaram a fazer o meu melhor e demonstraram interesse em tudo o que eu faço. Finally, I would like to thank for the financial support provided by UCIBIO, REQUIMTE and Fundação para a Ciência e Tecnologia (PTDC/BIA-PRO/109796/2009 and SFRH/BD/87878/2012). v vi Abstract Bacteria display an array of enzymes to detoxify reactive oxygen species that cause cell damage and death, such as the bacterial cytochrome c peroxidase (BCCP) that reduces H2O2 to water in the periplasm. The BCCPs studied up-to-date are soluble dihemic enzymes from non-pathogenic bacteria. This thesis focus on the trihemic BCCP from Escherichia coli (YhjA), and the dihemic BCCP from the obligate human pathogen Neisseria gonorrhoeae (NgBCCP). These two enzymes are membrane anchored thus, soluble recombinant proteins of their conserved globular domains were produced, purified and characterized biochemically and spectroscopically. Recombinant NgBCCP, a 38 kDa protein, forms a homodimer in the presence of calcium ions. It contains a high-potential E heme (+310 mV, pH 7.5) and a low-potential P heme (-190 mV/-300 mV, pH 7.5), the active site, with a unique high-spin EPR signal at low temperatures in the mixed-valence active form. NgBCCP has catalytic activity with ABTS2- (synthetic electron donor) and a Lipid- modified Azurin (LAz) as electron donors (low KM values 4.0 and 0.4 µM H2O2, respectively) which was dependent on reductive activation and calcium ions, and optimum at physiological pH (7.0) and temperature (37 ºC). LAz, identified as NgBCCP physiological electron donor, was capable of activating the enzyme. The NgBCCP/LAz electron transfer complex has a low binding affinity (micromolar range), and the interaction is dynamic and of a hydrophobic nature according to NMR, docking and preliminary calorimetry studies. The peroxidase activity was inhibited by exogenous ligands bound at the active site, such as azide, cyanide and imidazole, as demonstrated by spectroscopic, kinetic and structural analysis. The structure of NgBCCP was determined for the mixed-valenced and azide-inhibited form, and a catalytic mechanism for BCCPs was proposed based on the structural analysis of NgBCCP active site. The recombinant YhjA, a 50 kDa monomer, has a C-terminal domain homologous to dihemic BCCPs and a N-terminal (NT) domain. This domain was characterized for the first time, demonstrating that NT heme is His63/Met125 coordinated. The reduction potentials of P, NT and E hemes were determined: –170 mV, +133 mV and +210 mV, at pH 7.5, respectively. YhjA has quinol peroxidase activity in vitro (millimolar range KM values) using hydroquinone and menadiol (menaquinol analogue), as electron donors. Calcium ions were needed for maximum activity but not reductive activation, as P heme is always high-spin penta-coordinated. This property allowed to detect the formation of an intermediate radical species upon incubation with H2O2. Real Time PCR data showed that YhjA was expressed under anaerobic conditions, which agrees with the use of menaquinol in those conditions. Hence it was suggested a role in H2O2 detoxification when transitioning from anaerobic to aerobic environments. Keywords: bacterial peroxidases, oxidative stress response, Neisseria gonorrhoeae, Escherichia coli, electron transfer complex, catalytic mechanism. vii viii Resumo As bactérias dispõem de um conjunto de enzimas que removem as espécies reactivas de oxigénio que causam danos e morte celular, tais como a peroxidase de citocromo c bacteriana (BCCP) que reduz H2O2 a água no periplasma. As BCCPs estudadas até à data são enzimas di-hémicas solúveis de bactérias não patogénicas. Esta tese foca-se na BCCP tri-hémica de Escherichia coli (YhjA), e na BCCP di-hémica da bactéria patogénica Neisseria gonorrhoeae (NgBCCP). Estas duas enzimas estão ancoradas à membrana pelo que, proteínas recombinantes solúveis do domínio globular foram produzidas, purificadas e caracterizadas bioquimicamente e espectroscopicamente. A NgBCCP recombinante, uma proteína de 38 kDa, forma um homodímero na presença de iões de cálcio. Esta proteína contém o hemo E de alto potencial (+310 mV, pH 7.5) e o hemo P de baixo potencial (-190 mV/-300 mV, pH 7.5), o centro activo, que apresenta um sinal de spin alto único no espectro de EPR da forma de valência mista, a baixas temperaturas. A NgBCCP tem actividade catalítica com ABTS2- (dador de electrões sintético) e a azurina modificada por lípidos (LAz), como dadores de electrões (valores de KM de 4.0 e 0.4 µM H2O2, respectivamente). A actividade da NgBCCP é dependente de activação por redução e de iões de cálcio, e é óptima a valores de pH (7.0) e temperatura (37 ºC) fisiológicos. A LAz, identificada como o dador de electrões fisiológico de NgBCCP, é capaz de activar esta enzima. O complexo de transferência electrónica NgBCCP/LAz tem uma afinidade de ligação baixa (na ordem de micromolar) e tem uma interação dinâmica e de natureza hidrofóbica de acordo com os dados de NMR, “docking” e estudos preliminares de calorimetria. A análise espectroscópica, ensaios cinéticos e análise estrutural demonstraram que a actividade da NgBCCP é inibida pela ligação de ligandos exógenos ao centro activo, tais como a azida, cianeto e imidazol.
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