130875 20240201151019.0 doi 10.1016/j.bbabio.2019.148058 sideral 114136 ART-2019-114136 eng Perez-Amigot, Daniel Towards the competent conformation for catalysis in the ferredoxin-NADP(+) reductase from the Brucella ovis pathogen 2019 Brucella ovis encodes a bacterial subclass 1 ferredoxin-NADP(H) reductase (BoFPR) that, by similarity with other FPRs, is expected either to deliver electrons from NADPH to the redox-based metabolism and/or to oxidize NADPH to regulate the soxRS regulon that protects bacteria against oxidative damage. Such potential roles for the pathogen survival under infection conditions make of interest to understand and to act on the BoFPR mechanism. Here, we investigate the NADP(+)/H interaction and NADPH oxidation by hydride transfer (HT) to BoFPR. Crystal structures of BoFPR in free and in complex with NADP(+) hardly differ. The latter shows binding of the NADP(+) adenosine moiety, while its redox-reactive nicotinamide protrudes towards the solvent. Nonetheless, pre-steady-state kinetics show formation of a charge-transfer complex (CTC-1) prior to the hydride transfer, as well as conversion of CTC-1 into a second charge-transfer complex (CTC-2) concomitantly with the HT event. Thus, during catalysis nicotinamide and flavin reacting rings stack. Kinetic data also identify the HT itself as the rate limiting step in the reduction of BoFPR by NADPH, as well as product release limiting the overall reaction. Using all-atom molecular dynamics simulations with a thermal effect approach we are able to visualise a potential transient catalytically competent interaction of the reacting rings. Simulations indicate that the architecture of the FAD folded conformation in BoFPR might be key in catalysis, pointing to its adenine as an element to orient the reactive atoms in conformations competent for HT. info:eu-repo/grantAgreement/ES/DGA/E35-17R info:eu-repo/grantAgreement/ES/MINECO-AEI-FEDER/BIO2016-75183-P info:eu-repo/semantics/closedAccess All rights reserved http://www.europeana.eu/rights/rr-f/ 3.465 2019 BIOPHYSICS 18 / 71 = 0.254 2019 Q2 T1 BIOCHEMISTRY & MOLECULAR BIOLOGY 125 / 296 = 0.422 2019 Q2 T2 2.001 2019 Biochemistry 2019 Q1 Cell Biology 2019 Q1 Biophysics 2019 Q1 info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion Taleb, Víctor Universidad de Zaragoza (orcid)0000-0001-9224-5854 Boneta, Sergio Anoz-Carbonell, Ernesto Universidad de Zaragoza (orcid)0000-0002-6649-9153 Sebastian, María (orcid)0000-0001-7202-4587 Velazquez-Campoy, Adrián Universidad de Zaragoza (orcid)0000-0001-5702-4538 Polo, Víctor Universidad de Zaragoza (orcid)0000-0001-5823-7965 Martinez-Julvez, Marta Universidad de Zaragoza (orcid)0000-0001-9047-0046 Medina, Milagros Universidad de Zaragoza (orcid)0000-0001-8743-0182 2012 755 Universidad de Zaragoza Dpto. Química Física Área Química Física 1002 060 Universidad de Zaragoza Dpto. Bioq.Biolog.Mol. Celular Área Bioquímica y Biolog.Mole. 1860, 10 (2019), 148058 [12 pp.] Biochim. biophys. acta, Bioenerg. BIOCHIMICA ET BIOPHYSICA ACTA-BIOENERGETICS 0005-2728 2279438 http://zaguan.unizar.es/record/130875/files/texto_completo.pdf Versión publicada 2404734 http://zaguan.unizar.es/record/130875/files/texto_completo.jpg?subformat=icon icon Versión publicada oai:zaguan.unizar.es:130875 articulos driver 2024-02-01-14:36:49 ARTICLE