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

CATALANO DUPUY, D; RIAL, D; CECCARELLI, EDUARDO A.

Pinamar, Argentina

Congreso; 10th Congress Panamerican Association for Biochemistry and Molecular Biology; 2005

SAIB - PABMB

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 is a very poor NAD(H) oxidoreductase. In contrast, a number of redox compounds, can replace its natural substrates (ferredoxin or flavodoxin) as electron acceptors in vitro.  FNRs consist 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. Searching for evidences of the mentioned displacement we constructed a mutant FNR in which the Y308 is followed by a tail of nine amino acids, including four histidine residues. In the presence of a metal ion this added structure could fold itself and, as consequence, would partially impair the tyrosine residue movement. Kinetics studies of this mutant FNR showed a decline in enzyme catalytic efficiency either in the presence of Zn2+ or Co2+.  However, the enzyme affinity for NADP(H) was not changed under this conditions. Our results are the first experimental evidence that Y308 mobility is essential for obtaining a high enzyme catalytic efficiency.