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dc.contributor.author
Bortolotti, Ana  
dc.contributor.author
Sánchez Azqueta, Ana  
dc.contributor.author
Maya, Celia M.  
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Velázquez Campoy, Adrian  
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Hermoso, Juan A  
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Medina, Milagros  
dc.contributor.author
Cortez, Nestor Ricardo  
dc.date.available
2017-12-05T14:01:18Z  
dc.date.issued
2014-01  
dc.identifier.citation
Bortolotti, Ana; Sánchez Azqueta, Ana; Maya, Celia M.; Velázquez Campoy, Adrian; Hermoso, Juan A; et al.; The C-terminal extension of bacterial flavodoxin-reductases: Involvement in the hydride transfer mechanism from the coenzyme; Elsevier Science; Biochimica Et Biophysica Acta-bioenergetics; 1837; 1; 1-2014; 33-43  
dc.identifier.issn
0005-2728  
dc.identifier.uri
http://hdl.handle.net/11336/29689  
dc.description.abstract
To study the role of the mobile C-terminal extension present in bacterial class of plant type NADP(H):ferredoxin reductases during catalysis, we generated a series of mutants of the Rhodobacter capsulatus enzyme (RcFPR). Deletion of the six C-terminal amino acids beyond alanine 266 was combined with the replacement A266Y, emulating the structure present in plastidic versions of this flavoenzyme. Analysis of absorbance and fluorescence spectra suggests that deletion does not modify the general geometry of FAD itself, but increases exposure of the flavin to the solvent, prevents a productive geometry of FAD: NADP(H) complex and decreases the protein thermal stability. Although the replacement A266Y partially coats the isoalloxazine from solvent and slightly restores protein stability, this single change does not allow formation of active charge-transfer complexes commonly present in the wild-type FPR, probably due to restraints of C-terminus pliability. A proton exchange process is deduced from ITC measurements during coenzyme binding. All studied RcFPR variants display higher affinity for NADP+ than wild-type, evidencing the contribution of the C-terminus in tempering a non-productive strong (rigid) interaction with the coenzyme. The decreased catalytic rate parameters confirm that the hydride transfer from NADPH to the flavin ring is considerably hampered in the mutants. Although the involvement of the Cterminal extension from bacterial FPRs in stabilizing overall folding and bent-FAD geometry has been stated, the most relevant contributions to catalysis are modulation of coenzyme entrance and affinity, promotion of the optimal geometry of an active complex and supply of a proton acceptor acting during coenzyme binding  
dc.format
application/pdf  
dc.language.iso
eng  
dc.publisher
Elsevier Science  
dc.rights
info:eu-repo/semantics/openAccess  
dc.rights.uri
https://creativecommons.org/licenses/by-nc-sa/2.5/ar/  
dc.subject
Ferredoxin (Flavodoxin)-Nadp+ Reductase  
dc.subject
Catalytic Efficiency  
dc.subject
Crystal Structure  
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Hydride Transfer  
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Otras Ciencias Biológicas  
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Ciencias Biológicas  
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CIENCIAS NATURALES Y EXACTAS  
dc.title
The C-terminal extension of bacterial flavodoxin-reductases: Involvement in the hydride transfer mechanism from the coenzyme  
dc.type
info:eu-repo/semantics/article  
dc.type
info:ar-repo/semantics/artículo  
dc.type
info:eu-repo/semantics/publishedVersion  
dc.date.updated
2017-12-04T19:17:21Z  
dc.journal.volume
1837  
dc.journal.number
1  
dc.journal.pagination
33-43  
dc.journal.pais
Países Bajos  
dc.journal.ciudad
Amsterdam  
dc.description.fil
Fil: Bortolotti, Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina  
dc.description.fil
Fil: Sánchez Azqueta, Ana. Universidad de Zaragoza; España  
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Fil: Maya, Celia M.. Universidad de Sevilla; España  
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Fil: Velázquez Campoy, Adrian. Universidad de Zaragoza; España. Fundacion ARAID. Aragón; España  
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Fil: Hermoso, Juan A. Instituto de Química Física Rocasolano. Madrid; España  
dc.description.fil
Fil: Medina, Milagros. Universidad de Zaragoza; España  
dc.description.fil
Fil: Cortez, Nestor Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentina  
dc.journal.title
Biochimica Et Biophysica Acta-bioenergetics  
dc.relation.alternativeid
info:eu-repo/semantics/altIdentifier/doi/http://dx.doi.org/10.1016/j.bbabio.2013.08.008  
dc.relation.alternativeid
info:eu-repo/semantics/altIdentifier/url/http://www.sciencedirect.com/science/article/pii/S000527281300145