Study of the Cys-His bridge electron transfer pathway in a copper-containing nitrite reductase by site-directed mutagenesis, spectroscopic, and computational methods
- Autores
- Cristaldi, Julio César; Gómez, María C.; González, Pablo Javier; Ferroni, Felix Martín; Dalosto, Sergio Daniel; Rizzi, Alberto Claudio; Rivas, Maria Gabriela; Brondino, Carlos Dante
- Año de publicación
- 2018
- Idioma
- inglés
- Tipo de recurso
- artículo
- Estado
- versión publicada
- Descripción
- The Cys-His bridge as electron transfer conduit in the enzymatic catalysis of nitrite to nitric oxide by nitrite reductase from Sinorhizobium meliloti 2011 (SmNir) was evaluated by site-directed mutagenesis, steady state kinetic studies, UV-vis and EPR spectroscopic measurements as well as computational calculations. The kinetic, structural and spectroscopic properties of the His171Asp (H171D) and Cys172Asp (C172D) SmNir variants were compared with the wild type enzyme. Molecular properties of H171D and C172D indicate that these point mutations have not visible effects on the quaternary structure of SmNir. Both variants are catalytically incompetent using the physiological electron donor pseudoazurin, though C172D presents catalytic activity with the artificial electron donor methyl viologen (kcat =3.9(4) s-1) lower than that of wt SmNir (kcat =240(50) s-1). QM/MM calculations indicate that the lack of activity of H171D may be ascribed to the Nδ1H...OC hydrogen bond that partially shortcuts the T1-T2 bridging Cys-His covalent pathway. The role of the Nδ1H...OC hydrogen bond in the pH-dependent catalytic activity of wt SmNir is also analyzed by monitoring the T1 and T2 oxidation states at the end of the catalytic reaction of wt SmNir at pH6 and 10 by UV-vis and EPR spectroscopies. These data provide insight into how changes in Cys-His bridge interrupts the electron transfer between T1 and T2 and how the pH-dependent catalytic activity of the enzyme are related to pH-dependent structural modifications of the T1-T2 bridging chemical pathway.
Fil: Cristaldi, Julio César. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina
Fil: Gómez, María C.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina
Fil: González, Pablo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina
Fil: Ferroni, Felix Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina
Fil: Dalosto, Sergio Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Física del Litoral. Universidad Nacional del Litoral. Instituto de Física del Litoral; Argentina
Fil: Rizzi, Alberto Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina
Fil: Rivas, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina
Fil: Brondino, Carlos Dante. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina - Materia
-
COPPER
DENITRIFICATION
ELECTRON TRANSFER PATHWAY
ENZYME MECHANISM
NITRITE REDUCTASE
SINORHIZOBIUM MELILOTI 2011 - Nivel de accesibilidad
- acceso abierto
- Condiciones de uso
- https://creativecommons.org/licenses/by-nc-nd/2.5/ar/
- Repositorio
- Institución
- Consejo Nacional de Investigaciones Científicas y Técnicas
- OAI Identificador
- oai:ri.conicet.gov.ar:11336/37941
Ver los metadatos del registro completo
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oai:ri.conicet.gov.ar:11336/37941 |
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spelling |
Study of the Cys-His bridge electron transfer pathway in a copper-containing nitrite reductase by site-directed mutagenesis, spectroscopic, and computational methodsCristaldi, Julio CésarGómez, María C.González, Pablo JavierFerroni, Felix MartínDalosto, Sergio DanielRizzi, Alberto ClaudioRivas, Maria GabrielaBrondino, Carlos DanteCOPPERDENITRIFICATIONELECTRON TRANSFER PATHWAYENZYME MECHANISMNITRITE REDUCTASESINORHIZOBIUM MELILOTI 2011https://purl.org/becyt/ford/1.6https://purl.org/becyt/ford/1The Cys-His bridge as electron transfer conduit in the enzymatic catalysis of nitrite to nitric oxide by nitrite reductase from Sinorhizobium meliloti 2011 (SmNir) was evaluated by site-directed mutagenesis, steady state kinetic studies, UV-vis and EPR spectroscopic measurements as well as computational calculations. The kinetic, structural and spectroscopic properties of the His171Asp (H171D) and Cys172Asp (C172D) SmNir variants were compared with the wild type enzyme. Molecular properties of H171D and C172D indicate that these point mutations have not visible effects on the quaternary structure of SmNir. Both variants are catalytically incompetent using the physiological electron donor pseudoazurin, though C172D presents catalytic activity with the artificial electron donor methyl viologen (kcat =3.9(4) s-1) lower than that of wt SmNir (kcat =240(50) s-1). QM/MM calculations indicate that the lack of activity of H171D may be ascribed to the Nδ1H...OC hydrogen bond that partially shortcuts the T1-T2 bridging Cys-His covalent pathway. The role of the Nδ1H...OC hydrogen bond in the pH-dependent catalytic activity of wt SmNir is also analyzed by monitoring the T1 and T2 oxidation states at the end of the catalytic reaction of wt SmNir at pH6 and 10 by UV-vis and EPR spectroscopies. These data provide insight into how changes in Cys-His bridge interrupts the electron transfer between T1 and T2 and how the pH-dependent catalytic activity of the enzyme are related to pH-dependent structural modifications of the T1-T2 bridging chemical pathway.Fil: Cristaldi, Julio César. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; ArgentinaFil: Gómez, María C.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; ArgentinaFil: González, Pablo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; ArgentinaFil: Ferroni, Felix Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; ArgentinaFil: Dalosto, Sergio Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Física del Litoral. Universidad Nacional del Litoral. Instituto de Física del Litoral; ArgentinaFil: Rizzi, Alberto Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; ArgentinaFil: Rivas, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; ArgentinaFil: Brondino, Carlos Dante. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; ArgentinaElsevier Science2018-03info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttp://purl.org/coar/resource_type/c_6501info:ar-repo/semantics/articuloapplication/pdfapplication/pdfapplication/pdfhttp://hdl.handle.net/11336/37941Cristaldi, Julio César; Gómez, María C.; González, Pablo Javier; Ferroni, Felix Martín; Dalosto, Sergio Daniel; et al.; Study of the Cys-His bridge electron transfer pathway in a copper-containing nitrite reductase by site-directed mutagenesis, spectroscopic, and computational methods; Elsevier Science; Biochimica et Biophysica Acta- General Subjects; 1862; 3; 3-2018; 752-7600304-4165CONICET DigitalCONICETenginfo:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S0304416517303409info:eu-repo/semantics/altIdentifier/doi/10.1016/j.bbagen.2017.10.011info:eu-repo/semantics/openAccesshttps://creativecommons.org/licenses/by-nc-nd/2.5/ar/reponame:CONICET Digital (CONICET)instname:Consejo Nacional de Investigaciones Científicas y Técnicas2025-09-29T10:03:11Zoai:ri.conicet.gov.ar:11336/37941instacron:CONICETInstitucionalhttp://ri.conicet.gov.ar/Organismo científico-tecnológicoNo correspondehttp://ri.conicet.gov.ar/oai/requestdasensio@conicet.gov.ar; lcarlino@conicet.gov.arArgentinaNo correspondeNo correspondeNo correspondeopendoar:34982025-09-29 10:03:12.168CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicasfalse |
dc.title.none.fl_str_mv |
Study of the Cys-His bridge electron transfer pathway in a copper-containing nitrite reductase by site-directed mutagenesis, spectroscopic, and computational methods |
title |
Study of the Cys-His bridge electron transfer pathway in a copper-containing nitrite reductase by site-directed mutagenesis, spectroscopic, and computational methods |
spellingShingle |
Study of the Cys-His bridge electron transfer pathway in a copper-containing nitrite reductase by site-directed mutagenesis, spectroscopic, and computational methods Cristaldi, Julio César COPPER DENITRIFICATION ELECTRON TRANSFER PATHWAY ENZYME MECHANISM NITRITE REDUCTASE SINORHIZOBIUM MELILOTI 2011 |
title_short |
Study of the Cys-His bridge electron transfer pathway in a copper-containing nitrite reductase by site-directed mutagenesis, spectroscopic, and computational methods |
title_full |
Study of the Cys-His bridge electron transfer pathway in a copper-containing nitrite reductase by site-directed mutagenesis, spectroscopic, and computational methods |
title_fullStr |
Study of the Cys-His bridge electron transfer pathway in a copper-containing nitrite reductase by site-directed mutagenesis, spectroscopic, and computational methods |
title_full_unstemmed |
Study of the Cys-His bridge electron transfer pathway in a copper-containing nitrite reductase by site-directed mutagenesis, spectroscopic, and computational methods |
title_sort |
Study of the Cys-His bridge electron transfer pathway in a copper-containing nitrite reductase by site-directed mutagenesis, spectroscopic, and computational methods |
dc.creator.none.fl_str_mv |
Cristaldi, Julio César Gómez, María C. González, Pablo Javier Ferroni, Felix Martín Dalosto, Sergio Daniel Rizzi, Alberto Claudio Rivas, Maria Gabriela Brondino, Carlos Dante |
author |
Cristaldi, Julio César |
author_facet |
Cristaldi, Julio César Gómez, María C. González, Pablo Javier Ferroni, Felix Martín Dalosto, Sergio Daniel Rizzi, Alberto Claudio Rivas, Maria Gabriela Brondino, Carlos Dante |
author_role |
author |
author2 |
Gómez, María C. González, Pablo Javier Ferroni, Felix Martín Dalosto, Sergio Daniel Rizzi, Alberto Claudio Rivas, Maria Gabriela Brondino, Carlos Dante |
author2_role |
author author author author author author author |
dc.subject.none.fl_str_mv |
COPPER DENITRIFICATION ELECTRON TRANSFER PATHWAY ENZYME MECHANISM NITRITE REDUCTASE SINORHIZOBIUM MELILOTI 2011 |
topic |
COPPER DENITRIFICATION ELECTRON TRANSFER PATHWAY ENZYME MECHANISM NITRITE REDUCTASE SINORHIZOBIUM MELILOTI 2011 |
purl_subject.fl_str_mv |
https://purl.org/becyt/ford/1.6 https://purl.org/becyt/ford/1 |
dc.description.none.fl_txt_mv |
The Cys-His bridge as electron transfer conduit in the enzymatic catalysis of nitrite to nitric oxide by nitrite reductase from Sinorhizobium meliloti 2011 (SmNir) was evaluated by site-directed mutagenesis, steady state kinetic studies, UV-vis and EPR spectroscopic measurements as well as computational calculations. The kinetic, structural and spectroscopic properties of the His171Asp (H171D) and Cys172Asp (C172D) SmNir variants were compared with the wild type enzyme. Molecular properties of H171D and C172D indicate that these point mutations have not visible effects on the quaternary structure of SmNir. Both variants are catalytically incompetent using the physiological electron donor pseudoazurin, though C172D presents catalytic activity with the artificial electron donor methyl viologen (kcat =3.9(4) s-1) lower than that of wt SmNir (kcat =240(50) s-1). QM/MM calculations indicate that the lack of activity of H171D may be ascribed to the Nδ1H...OC hydrogen bond that partially shortcuts the T1-T2 bridging Cys-His covalent pathway. The role of the Nδ1H...OC hydrogen bond in the pH-dependent catalytic activity of wt SmNir is also analyzed by monitoring the T1 and T2 oxidation states at the end of the catalytic reaction of wt SmNir at pH6 and 10 by UV-vis and EPR spectroscopies. These data provide insight into how changes in Cys-His bridge interrupts the electron transfer between T1 and T2 and how the pH-dependent catalytic activity of the enzyme are related to pH-dependent structural modifications of the T1-T2 bridging chemical pathway. Fil: Cristaldi, Julio César. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina Fil: Gómez, María C.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina Fil: González, Pablo Javier. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina Fil: Ferroni, Felix Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina Fil: Dalosto, Sergio Daniel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Física del Litoral. Universidad Nacional del Litoral. Instituto de Física del Litoral; Argentina Fil: Rizzi, Alberto Claudio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina Fil: Rivas, Maria Gabriela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina Fil: Brondino, Carlos Dante. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe; Argentina. Universidad Nacional del Litoral. Facultad de Bioquímica y Ciencias Biológicas. Departamento de Física; Argentina |
description |
The Cys-His bridge as electron transfer conduit in the enzymatic catalysis of nitrite to nitric oxide by nitrite reductase from Sinorhizobium meliloti 2011 (SmNir) was evaluated by site-directed mutagenesis, steady state kinetic studies, UV-vis and EPR spectroscopic measurements as well as computational calculations. The kinetic, structural and spectroscopic properties of the His171Asp (H171D) and Cys172Asp (C172D) SmNir variants were compared with the wild type enzyme. Molecular properties of H171D and C172D indicate that these point mutations have not visible effects on the quaternary structure of SmNir. Both variants are catalytically incompetent using the physiological electron donor pseudoazurin, though C172D presents catalytic activity with the artificial electron donor methyl viologen (kcat =3.9(4) s-1) lower than that of wt SmNir (kcat =240(50) s-1). QM/MM calculations indicate that the lack of activity of H171D may be ascribed to the Nδ1H...OC hydrogen bond that partially shortcuts the T1-T2 bridging Cys-His covalent pathway. The role of the Nδ1H...OC hydrogen bond in the pH-dependent catalytic activity of wt SmNir is also analyzed by monitoring the T1 and T2 oxidation states at the end of the catalytic reaction of wt SmNir at pH6 and 10 by UV-vis and EPR spectroscopies. These data provide insight into how changes in Cys-His bridge interrupts the electron transfer between T1 and T2 and how the pH-dependent catalytic activity of the enzyme are related to pH-dependent structural modifications of the T1-T2 bridging chemical pathway. |
publishDate |
2018 |
dc.date.none.fl_str_mv |
2018-03 |
dc.type.none.fl_str_mv |
info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion http://purl.org/coar/resource_type/c_6501 info:ar-repo/semantics/articulo |
format |
article |
status_str |
publishedVersion |
dc.identifier.none.fl_str_mv |
http://hdl.handle.net/11336/37941 Cristaldi, Julio César; Gómez, María C.; González, Pablo Javier; Ferroni, Felix Martín; Dalosto, Sergio Daniel; et al.; Study of the Cys-His bridge electron transfer pathway in a copper-containing nitrite reductase by site-directed mutagenesis, spectroscopic, and computational methods; Elsevier Science; Biochimica et Biophysica Acta- General Subjects; 1862; 3; 3-2018; 752-760 0304-4165 CONICET Digital CONICET |
url |
http://hdl.handle.net/11336/37941 |
identifier_str_mv |
Cristaldi, Julio César; Gómez, María C.; González, Pablo Javier; Ferroni, Felix Martín; Dalosto, Sergio Daniel; et al.; Study of the Cys-His bridge electron transfer pathway in a copper-containing nitrite reductase by site-directed mutagenesis, spectroscopic, and computational methods; Elsevier Science; Biochimica et Biophysica Acta- General Subjects; 1862; 3; 3-2018; 752-760 0304-4165 CONICET Digital CONICET |
dc.language.none.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
info:eu-repo/semantics/altIdentifier/url/https://www.sciencedirect.com/science/article/pii/S0304416517303409 info:eu-repo/semantics/altIdentifier/doi/10.1016/j.bbagen.2017.10.011 |
dc.rights.none.fl_str_mv |
info:eu-repo/semantics/openAccess https://creativecommons.org/licenses/by-nc-nd/2.5/ar/ |
eu_rights_str_mv |
openAccess |
rights_invalid_str_mv |
https://creativecommons.org/licenses/by-nc-nd/2.5/ar/ |
dc.format.none.fl_str_mv |
application/pdf application/pdf application/pdf |
dc.publisher.none.fl_str_mv |
Elsevier Science |
publisher.none.fl_str_mv |
Elsevier Science |
dc.source.none.fl_str_mv |
reponame:CONICET Digital (CONICET) instname:Consejo Nacional de Investigaciones Científicas y Técnicas |
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Consejo Nacional de Investigaciones Científicas y Técnicas |
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CONICET Digital (CONICET) - Consejo Nacional de Investigaciones Científicas y Técnicas |
repository.mail.fl_str_mv |
dasensio@conicet.gov.ar; lcarlino@conicet.gov.ar |
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13.070432 |