Modification of its C-terminus in a bacterial ferredoxin/flavodoxin NADP+reductase modulates spectroscopic and photophysical properties of FAD

L. VALLE; I. ABATEDAGA; F.E. MORÁN VIEYRA; A. BORTOLOTTI; N. CORTEZ; C.D. BORSARELLI

Córdoba

Congreso; 16th INTERNATIONAL CONGRESS ON PHOTOBIOLOGY; 2014

International Union of Photobiology

The photosynthetic bacterium Rhodobacter capsulatus contains a single (flavodoxin)-NADP(H) oxidoreductase (RcFPR) that catalyzes the electron transfer from NADP(H) to the flavodoxin nifF for nitrogenase reduction [1,2]. The plant type of these reductases presents a C-terminal Tyr residue involved in catalytic mechanism and stabilizing the re-face of the isoalloxazine ring of FAD. Differently, bacterial class FPRs bear a Phe or Ala residue instead of terminal Tyr, and a ending C-terminal extension = FVGEGI, which also participates in NADP(H) binding and hydride transfer mechanism [1, 2]. In this work, a site-directed mutagenesis approach on RcFPR was performed, producing three different mutants: A266Y, A266 and A266Y, where  represents mutants without the terminal peptide FVGEGI, and its effect on the photophysical properties of FAD was evaluated. UV-vis absorption, and steady-state and dynamic fluorescence spectroscopy results indicated that the changes on composition of the C-terminal region resulted in the distortion of the appropriate distance and orientation of the isoalloxazine ring of FAD with the highly conserved Tyr66, producing efficient ?switching-off? of the ultra-fast CT-quenching of FAD observed in the native enzyme. Contrary to the native enzyme, in the the modified enzymes, the isoalloxazine ring of FAD is located in a much less rigid nanospace of the enzyme, allowing more segmental motion of the isoalloxazine ring. The addition of extra Try266 did not increase the CT-quenching of FAD, suggesting that the Tyr266 does not stack the re-face of the isoalloxazine ring of the cofactor, as occur in plants FNRs [1]. Laser flash photolysis and O2-uptake studies demonstrated that in modified RcFPR, FAD forms long-lived excited triplet state, which is quenched by O2 producing reactive oygen species that produce cofactor and protein degradation. These results reflect the delicate balance between enzyme structural conformation and functionality, since in native RcFPR where the isoalloxazine ring of FAD is tightly bound in the closer nanospace of Tyr66, the ultra-fast CT quenching is produced avoiding the formation of reactive triplet excited of the flavin, preserving the enzyme of self-photodegradation and favoring its enzymatic function involving the ground-state of FAD.