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Article

Nanomechanical Study of Enzyme: Coenzyme Complexes: Bipartite Sites in Plastidic Ferredoxin-NADP+ Reductase for the Interaction with NADP+

1
Instituto de Nanociencia y Materiales de Aragón (INMA), CSIC-Universidad de Zaragoza, 50009 Zaragoza, Spain
2
Laboratorio de Microscopías Avanzadas (LMA), Universidad de Zaragoza, 50018 Zaragoza, Spain
3
Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Instituto de Biocomputación y Física de Sistemas Complejos (BIFI) (GBsC-CSIC Joint Unit), Universidad de Zaragoza, 50018 Zaragoza, Spain
4
Fundación ARAID, 50018 Zaragoza, Spain
*
Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Academic Editors: Daisuke Seo and Narimantas K. Cenas
Antioxidants 2022, 11(3), 537; https://doi.org/10.3390/antiox11030537
Received: 9 February 2022 / Revised: 7 March 2022 / Accepted: 8 March 2022 / Published: 11 March 2022
Plastidic ferredoxin-NADP+ reductase (FNR) transfers two electrons from two ferredoxin or flavodoxin molecules to NADP+, generating NADPH. The forces holding the Anabaena FNR:NADP+ complex were analyzed by dynamic force spectroscopy, using WT FNR and three C-terminal Y303 variants, Y303S, Y303F, and Y303W. FNR was covalently immobilized on mica and NADP+ attached to AFM tips. Force–distance curves were collected for different loading rates and specific unbinding forces were analyzed under the Bell–Evans model to obtain the mechanostability parameters associated with the dissociation processes. The WT FNR:NADP+ complex presented a higher mechanical stability than that reported for the complexes with protein partners, corroborating the stronger affinity of FNR for NADP+. The Y303 mutation induced changes in the FNR:NADP+ interaction mechanical stability. NADP+ dissociated from WT and Y303W in a single event related to the release of the adenine moiety of the coenzyme. However, two events described the Y303S:NADP+ dissociation that was also a more durable complex due to the strong binding of the nicotinamide moiety of NADP+ to the catalytic site. Finally, Y303F shows intermediate behavior. Therefore, Y303, reported as crucial for achieving catalytically competent active site geometry, also regulates the concerted dissociation of the bipartite nucleotide moieties of the coenzyme. View Full-Text
Keywords: ferredoxin NADP+ reductase; atomic force microscopy; dynamic force spectroscopy; NADP+; protein–ligand (substrate) interactions; single-molecule methods; flavoproteins; functionalization; nanomechanics; mechanical stability ferredoxin NADP+ reductase; atomic force microscopy; dynamic force spectroscopy; NADP+; protein–ligand (substrate) interactions; single-molecule methods; flavoproteins; functionalization; nanomechanics; mechanical stability
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MDPI and ACS Style

Pérez-Domínguez, S.; Caballero-Mancebo, S.; Marcuello, C.; Martínez-Júlvez, M.; Medina, M.; Lostao, A. Nanomechanical Study of Enzyme: Coenzyme Complexes: Bipartite Sites in Plastidic Ferredoxin-NADP+ Reductase for the Interaction with NADP+. Antioxidants 2022, 11, 537. https://doi.org/10.3390/antiox11030537

AMA Style

Pérez-Domínguez S, Caballero-Mancebo S, Marcuello C, Martínez-Júlvez M, Medina M, Lostao A. Nanomechanical Study of Enzyme: Coenzyme Complexes: Bipartite Sites in Plastidic Ferredoxin-NADP+ Reductase for the Interaction with NADP+. Antioxidants. 2022; 11(3):537. https://doi.org/10.3390/antiox11030537

Chicago/Turabian Style

Pérez-Domínguez, Sandra, Silvia Caballero-Mancebo, Carlos Marcuello, Marta Martínez-Júlvez, Milagros Medina, and Anabel Lostao. 2022. "Nanomechanical Study of Enzyme: Coenzyme Complexes: Bipartite Sites in Plastidic Ferredoxin-NADP+ Reductase for the Interaction with NADP+" Antioxidants 11, no. 3: 537. https://doi.org/10.3390/antiox11030537

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