C-terminus mutation effects on spectroscopic properties of Rhodobacter capsulatus ferredoxin/flavodoxin NADP reductase

LORENA VALLE; INÉS ABATEDAGA; FAUSTINO MORÁN VIEYRA; ANA BORTOLOTTI; CLAUDIO BORSARELLI; NÉSTOR CORTEZ

Tucumán

Congreso; 1. XLI Reunión Anual de la Sociedad Argentina de Biofísica; 2012

The photosynthetic bacterium Rhodobacter capsulatus contains a single (flavodoxin)-NADP(H) oxidoreductase (RcFPR) that catalyzes electron transfer from NADP(H) to the flavodoxin nifF for nitrogenase reduction. The X-ray crystallography of the enzyme showed two domains harboring the FAD and NADP(H). Plastidic class of 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 carry a Phe or Ala instead terminal Tyr, and a C-terminal extension -FVGEGI, which also participates in NADP(H) binding and hydride transfer mechanisms. A site-directed mutagenesis approach on RcFPR was performed, producing three different mutants: A266Y, A266D and A266YD, which modifies both structural and catalytic properties . In this work, the UV-Vis absorption and fluorescence properties of FAD in the mutant series was analyzed. The results indicate the role of C-terminal extension -FVGEGI in the modulation of FAD exposure and stability in RcFPR.