Towards the competent conformation for catalysis in the ferredoxin-NADP+ reductase from the Brucella ovis pathogen

Publication date: Available online 5 August 2019Source: Biochimica et Biophysica Acta (BBA) - BioenergeticsAuthor(s): Daniel Pérez-Amigot, Víctor Taleb, Sergio Boneta, Ernesto Anoz-Carbonell, María Sebastián, Adrián Velázquez-Campoy, Víctor Polo, Marta Martínez-Júlvez, Milagros MedinaAbstractBrucella 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 r...
Source: Biochimica et Biophysica Acta (BBA) Bioenergetics - Category: Biochemistry Source Type: research