Overexpression and purification of Treponema pallidum rubredoxin; kinetic evidence for a superoxide-mediated electron transfer with the superoxide reductase neelaredoxin

Detalhes bibliográficos
Autor(a) principal: Auchère, Françoise
Data de Publicação: 2004
Outros Autores: Sikkink, Robert, Cordas, Cristina, Raleiras, Patrícia, Tavares, Pedro, Moura, Isabel, Moura, José J. G.
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
Texto Completo: http://hdl.handle.net/10362/8712
Resumo: J Biol Inorg Chem (2004) 9: 839–849 DOI 10.1007/s00775-004-0584-6
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spelling Overexpression and purification of Treponema pallidum rubredoxin; kinetic evidence for a superoxide-mediated electron transfer with the superoxide reductase neelaredoxinElectron transferNeelaredoxinRubredoxinSuperoxide reductaseTreponema pallidumJ Biol Inorg Chem (2004) 9: 839–849 DOI 10.1007/s00775-004-0584-6Superoxide reductases are a class of non-haem iron enzymes which catalyse the monovalent reduction of the superoxide anion O2- into hydrogen peroxide and water. Treponema pallidum (Tp), the syphilis spirochete, expresses the gene for a superoxide reductase called neelaredoxin, having the iron protein rubredoxin as the putative electron donor necessary to complete the catalytic cycle. In this work, we present the first cloning, overexpression in Escherichia coli and purification of the Tp rubredoxin. Spectroscopic characterization of this 6 kDa protein allowed us to calculate the molar absorption coefficient of the 490 nm feature of ferric iron, epsilon=6.9+/-0.4 mM(-1) cm(-1). Moreover, the midpoint potential of Tp rubredoxin, determined using a glassy carbon electrode, was -76+/-5 mV. Reduced rubredoxin can be efficiently reoxidized upon addition of Na(2)IrCl(6)-oxidized neelaredoxin, in agreement with a direct electron transfer between the two proteins, with a stoichiometry of the electron transfer reaction of one molecule of oxidized rubredoxin per one molecule of neelaredoxin. In addition, in presence of a steady-state concentration of superoxide anion, the physiological substrate of neelaredoxin, reoxidation of rubredoxin was also observed in presence of catalytic amounts of superoxide reductase, and the rate of rubredoxin reoxidation was shown to be proportional to the concentration of neelaredoxin, in agreement with a bimolecular reaction, with a calculated k(app)=180 min(-1). Interestingly, similar experiments performed with a rubredoxin from the sulfate-reducing bacteria Desulfovibrio vulgaris resulted in a much lower value of k(app)=4.5 min(-1). Altogether, these results demonstrated the existence for a superoxide-mediated electron transfer between rubredoxin and neelaredoxin and confirmed the physiological character of this electron transfer reaction.SpringerRUNAuchère, FrançoiseSikkink, RobertCordas, CristinaRaleiras, PatríciaTavares, PedroMoura, IsabelMoura, José J. G.2013-02-06T12:04:50Z20042004-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/10362/8712eng0949-8257info:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2024-03-11T03:41:36Zoai:run.unl.pt:10362/8712Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-20T03:18:23.608750Repositório 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 Overexpression and purification of Treponema pallidum rubredoxin; kinetic evidence for a superoxide-mediated electron transfer with the superoxide reductase neelaredoxin
title Overexpression and purification of Treponema pallidum rubredoxin; kinetic evidence for a superoxide-mediated electron transfer with the superoxide reductase neelaredoxin
spellingShingle Overexpression and purification of Treponema pallidum rubredoxin; kinetic evidence for a superoxide-mediated electron transfer with the superoxide reductase neelaredoxin
Auchère, Françoise
Electron transfer
Neelaredoxin
Rubredoxin
Superoxide reductase
Treponema pallidum
title_short Overexpression and purification of Treponema pallidum rubredoxin; kinetic evidence for a superoxide-mediated electron transfer with the superoxide reductase neelaredoxin
title_full Overexpression and purification of Treponema pallidum rubredoxin; kinetic evidence for a superoxide-mediated electron transfer with the superoxide reductase neelaredoxin
title_fullStr Overexpression and purification of Treponema pallidum rubredoxin; kinetic evidence for a superoxide-mediated electron transfer with the superoxide reductase neelaredoxin
title_full_unstemmed Overexpression and purification of Treponema pallidum rubredoxin; kinetic evidence for a superoxide-mediated electron transfer with the superoxide reductase neelaredoxin
title_sort Overexpression and purification of Treponema pallidum rubredoxin; kinetic evidence for a superoxide-mediated electron transfer with the superoxide reductase neelaredoxin
author Auchère, Françoise
author_facet Auchère, Françoise
Sikkink, Robert
Cordas, Cristina
Raleiras, Patrícia
Tavares, Pedro
Moura, Isabel
Moura, José J. G.
author_role author
author2 Sikkink, Robert
Cordas, Cristina
Raleiras, Patrícia
Tavares, Pedro
Moura, Isabel
Moura, José J. G.
author2_role author
author
author
author
author
author
dc.contributor.none.fl_str_mv RUN
dc.contributor.author.fl_str_mv Auchère, Françoise
Sikkink, Robert
Cordas, Cristina
Raleiras, Patrícia
Tavares, Pedro
Moura, Isabel
Moura, José J. G.
dc.subject.por.fl_str_mv Electron transfer
Neelaredoxin
Rubredoxin
Superoxide reductase
Treponema pallidum
topic Electron transfer
Neelaredoxin
Rubredoxin
Superoxide reductase
Treponema pallidum
description J Biol Inorg Chem (2004) 9: 839–849 DOI 10.1007/s00775-004-0584-6
publishDate 2004
dc.date.none.fl_str_mv 2004
2004-01-01T00:00:00Z
2013-02-06T12:04:50Z
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
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status_str publishedVersion
dc.identifier.uri.fl_str_mv http://hdl.handle.net/10362/8712
url http://hdl.handle.net/10362/8712
dc.language.iso.fl_str_mv eng
language eng
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dc.publisher.none.fl_str_mv Springer
publisher.none.fl_str_mv Springer
dc.source.none.fl_str_mv reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
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repository.name.fl_str_mv Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação
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