Involvement of ferredoxin-NA DP+ reductase FAD and NA DP+/H binding domains for optimal catalytic complex formation and hydride transfer efficiency

SÁNCHEZ-AZQUETA, A.; MUSUMECI, M. A.; CECCARELLI, E. A.; MEDINA, M

Zaragoza

Congreso; IV Spanish Portuguese Biophysical Congress; 2010

Portuguese and Spanish Biophysical Societies

In photosynthetic organisms, the enzyme responsible for NADPH production is ferredoxin-NADP+ reductase (FNR). This FAD-containing reductase takes two electrons from two reducedferredoxin (Fd) molecules, and transfers them to NADP+ in a single hydride transfer step. Thislater process has been reported to occur through the formation of two intermediate chargetransfer complexes species: FNRox-NADPH and FNRred-NADP+ [1]. Several FNR regions havebeen proposed as crucial for optimal FAD and NADP+ orientation during the hydride transferevent. Thus, Y79, S59 and the 102-110 loop (Anabaena FNR numbering) of the FAD bindingregion, are highly conserved among plastidic FNRs and apparently must contribute to FADallocation in the FAD-binding site. Therefore, these residues are expected to modulate theflavin oxido-reduction properties and might also contribute to the hydride transfer efficiency.In the NADP+ binding module, recent structural studies suggest that while E267 and E268might contribute to the displacement of the aromatic terminal tyrosine during the catalyticevent, the volume of residue C261 (C266 of pea FNR) is critical for the formation of a competentcatalytic complex [2]. In order to establish the role of all these residues in the hydridetransfer event we have produced either Anabaena or pea FNR site-directed mutants at thesepositions. The oxido-reduction properties of these variants, as well as their efficiency in formingthe intermediate charge transfer species and in hydride transfer during the reactionwith the coenzyme are here reported.[1] Tejero J, Peregrina JR, Martínez-Júlvez M, Gutiérrez A, Gómez-Moreno C, Scrutton NS, Medina M(2007) Arch. Biochem. Biophys. 459(1), 79-90.[2] Musumeci MA, Arakaki AK, Rial DV, Catalano-Dupuy DL, Ceccarelli EA (2008) FEBS J. 275(6), 1350-66.