SPECTROSCOPIC ANALYSIS OF THE INTERACTION BETWEEN NADP(H) AND A BACTERIAL FERREDOXIN-NADP REDUCTASE

BORTOLOTTI, A.; DUMIT, V.; GOÑI RASIA, G.; MEDINA, M.; CARRILLO, N.; CORTEZ, N.

Rosario (Argentina)

Congreso; XLII Reunión Anual de la Sociedad Argentina de investigación en Bioquímica y Biología Molecular SAIB; 2006

SAIB Sociedad Argentina de investigación en Bioquímica y Biología Molecular

Ferredoxin-NADP+ reductases are enzymes harbouring one molecule of noncovalently bound FAD. These flavoproteins (FNR/FPR) catalyse reversible reactions between obligatory one-electron carriers and a two-electron donors/acceptors. Even though members of the FNR/FPR superfamily (plant-type) exhibit a conserved structure, sequence analysis reveals two clusters, each one with a typical FAD conformation. This raises the distinction between FNRs in a plastidic class, characterised by extended FAD conformation and high catalytic efficiency, and a bacterial FPR class displaying a folded FAD molecule and low turnover rates. The interaction between Rhodobacter capsulatus FPR and NADP+ was studied by differential spectroscopy. Modification of the flavin absortion spectrum results from the binding of the nucleotide to the oxidised enzyme. These outcomes bear a resemblance to those reported for plastidic FNRs, suggesting that the interaction between FAD molecule and the nucleotidic partner is similar for both types of flavoproteins, irrespective of FAD conformation. Stopped-flow kinetics under anaerobic conditions was used to study the reaction between oxidised FPR and NADPH. The decrease in flavin absortion is related to its reduction by NADPH, in agreement with results previously obtained for the plastidic enzyme. We also detected two charge-transfer intermediates of the whole reactionRhodobacter capsulatus FPR and NADP+ was studied by differential spectroscopy. Modification of the flavin absortion spectrum results from the binding of the nucleotide to the oxidised enzyme. These outcomes bear a resemblance to those reported for plastidic FNRs, suggesting that the interaction between FAD molecule and the nucleotidic partner is similar for both types of flavoproteins, irrespective of FAD conformation. Stopped-flow kinetics under anaerobic conditions was used to study the reaction between oxidised FPR and NADPH. The decrease in flavin absortion is related to its reduction by NADPH, in agreement with results previously obtained for the plastidic enzyme. We also detected two charge-transfer intermediates of the whole reaction