Swapping FAD Binding Motifs Between Plastidic and Bacterial Ferredoxin-NADP(H) Reductases.

MATIAS A. MUSUMECI; HORACIO BOTTI; ALEJANDRO BUSCHIAZZO; EDUARDO A. CECCARELLI

BIOCHEMISTRY

AMER CHEMICAL SOC

Lugar: Washington D.C.; Año: 2010

0006-2960

Plant-type ferredoxin-NADP(H) reductases (FNRs) group in two classes, plastidic with an extended FAD conformation and high catalytic rates, and bacterial with a folded flavin nucleotide and low turnover rates. The beta-hairpin between amino acids 112 and 124 from pea FNR and the carboxy-terminal tryptophan of the Escherichia coli ferredoxin- NADP(H) reductase (FPR), suggested as responsible for the FAD differential conformation, were mutually exchanged. Pea FNR lacking the beta-hairpin was unable to fold properly. The inclusion of an extra tryptophan residue at the carboxy terminus, emulating the bacterial FPR, recovered the protein folding, decreased the affinity for FAD and reduced the diaphorase and ferredoxin-dependent cytochrome c reductase activities to 67% and 44% respectively. The sole inclusion of a carboxy-terminal tryptophan in pea FNR reduced the cytochrome c reductase activity without altering the FAD binding affinity. The insertion of the beta-hairpin into the corresponding position in FPR resulted in increased FAD affinity. Finally, a chimeric FPR in which the beta-hairpin was inserted and the carboxy-terminal tryptophan was simultaneously deleted showed a 2.6 and 8.6-fold increase in kcat and catalytic efficiency for the NADPH diaphorase activity respectively. This variant retained 10% of the cytochrome c reductase activity with ferredoxina and was unable to use flavodoxin as substrate. Crystallographic structures of the chimeras showed no significant changes in their overall structure, although alterations in the FAD conformations were observed. This report highlights the role of the FAD moiety conformation and the structural determinants involved in stabilizing it, ultimately modulating the functional output of FNRs.