The involvement of C-terminus in the catalytic mechanism of a bacterial ferredoxin-NADP+ reductase

ANA BORTOLOTTI; ANA SANCHEZ; MILAGROS MEDINA; NÉSTOR CORTEZ

Salta-Argentina

Congreso; 3rd Latin American Protein Society Meeting (LAPSM); 2010

Ferredoxin-NADP+ reductases (FPRs) are flavoenzymes that catalyze reversible reactions between obligatory one-electron carriers and two-electron donors/acceptors. Flavoenzymes involved in oxygenic photosynthesis display a high catalytic efficiency, carry an extended FAD conformation and an invariant tyrosine residue at the C-terminus facing the isoallozacine ring. Instead, enzymes present in eubacteria bind FAD in a bent conformation and have longer C-terminal tails with an aromatic or aliphatic amino acid facing the flavin. In the Rhodobacter capsulatus reductase, the binding of NADP(H) leads the displacement of the C-terminal tail of the protein to accommodate the nicotinamide ring allowing hydride transfer. We evaluate herein the contribution of the terminal residues A267 to I272 in the coenzyme binding during the catalytic process. The flavoenzyme mutants A266Y, A266D and A266YD  were generated. Although FAD was stably bound to all the studied mutants, the level of cofactor exposition reaches values equivalent to free FAD in solution and presented a decrease in thermal stability. C-teminus mutated FPRs displayed similar affinity to nucleotide coenzyme, almost 20-fold higher than wild type enzyme. A six fold decrease of activity resulted from the A266Y mutation, but a still higher fall of catalytic rates corresponded to the A266YD and A266D forms. At difference with the wild type enzyme, deletion mutants do not reveal the presence of charge-transfer complexes during the catalytic process supporting the involvement of the C-terminal tail structure in the rate and the mode of hydride transfer in bacterial ferredoxin-NADP+ reductases.