Modulation of the pea Ferredoxin-NADP+ reductase activity by a metal site

CATALANO DUPUY, DANIELA L.; RIAL, DANIELA V.; CECCARELLI, EDUARDO A.

Pinamar, Buenos Aires, ARGENTINA

Congreso; XLI Reunión Anual de SAIB; 2005

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Ferredoxin-NADP(H) reductases (FNR) are ubiquitous flavoenzymes that participate in a wide range of redox metabolic pathways in a variety of organisms. The plant-type reductase displays strong preference for NADP(H) and a high catalytic efficiency. The enzyme consists of two domains, one involved in the binding of the prosthetic group FAD and the other responsible for binding of NADP+. Two tyrosine residues lie close to each side of the isoalloxazine. The highly conserved Y308 in pea FNR is stacked near parallel to the re-face of the flavin and should be displaced by the nicotinamide ring of NADP(H) for productive binding to the enzyme. In order to find an experimental evidence of the mentioned displacement we constructed a mutant FNR in which the Y308 is followed by a tail of nine aminoacids, including four histidine residues. In the presence of a metal ion this added structure could fold itself and, as consequence, would impair the tyrosine residue movement. Kinetics studies of this mutant FNR showed a notorious decline in enzyme catalytic efficiency either in the presence of Zn2+ or Co2+. Spectrophotometric analysis indicated that the enzyme affinity for NADP(H) do not change under this condition and that the FNR is properly folded. Our results are the first experimental evidence that Y308 mobility is essential for obtaining a high enzyme catalytic efficiency.