Impact on the photophysical properties of cofactor FAD by C-terminus domain mutation in Rhodobacter capsulatus ferredoxin/flavodoxin NADP reductase

MORÁN-VIEYRA, FAUSTINO E.; VALLE, LORENA; ABATEDAGA, INÉS; BORTOLOTTI, ANA; CORTEZ, NÉSTOR; BORSARELLI, CLAUDIO

Córdoba

Congreso; II Reunión del Grupo Argentino de Fotobiología-GRAFOB; 2013

GRAFOB-Grupo Argentino de Fotobiología

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. 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 Δ = -FVGEGI, which also participates in NADP(H) binding and hydride transfer mechanism. A site-directed mutagenesis approach on RcFPR was performed, producing three different mutants: A266Y, A266Δ and A266YΔ, where Δ represents mutants without the terminal peptide FVGEGI. In this work, the photophysical properties of FAD in the above mutant series were analyzed by steady-state and time-resolved UV-Vis absorption and emission spectroscopies, and the role of both Ala-Tyr and ?FVGEGI modifications are discussed. Although catalytic activity is present in all cases, in aerated solutions of A266Δ a 10-fold higher fluorescence quantum yield was observed as compared with FAD in buffer, but with a similar quantum yield of excited triplet state (ΦT ≈ 0.35). This is totally contrary to the wild type protein, which almost does not fluoresce and not show excited triplet state of FAD. However, for the mutant A266Δ, the formation of singlet molecular oxygen was not detected. These results indicate how delicate is the tuning of photophysical properties of flavin cofactors in the protein cavity.