Biomedical subjects

Susana Frago

Publications and source records attributed to Susana Frago.

4 recordsLinked to original sources

PubMed2005

The ferredoxin-NADP(H) reductase from Rhodobacter capsulatus: molecular structure and catalytic mechanism.

The photosynthetic bacterium Rhodobacter capsulatus contains a ferredoxin (flavodoxin)-NADP(H) oxidoreductase (FPR) that catalyzes electron transfer between NADP(H) and ferredoxin or flavodoxin. The structure of the enzyme, determined by X-ray crystallography, contains two domains harboring the FAD and NADP(H) binding sites, as is typical of the FPR structural family. The FAD molecule is in a hairpin conformation in which stacking interactions can be established between the dimethylisoalloxazine and adenine moieties. The midpoint redox potentials of the various transitions undergone by R. capsulatus FPR were similar to those reported for their counterparts involved in oxygenic photosynthesis, but its catalytic activity is orders of magnitude lower (1-2 s(-)(1) versus 200-500 s(-)(1)) as is true for most of its prokaryotic homologues. To identify the mechanistic basis for the slow turnover in the bacterial enzymes, we dissected the R. capsulatus FPR reaction into hydride transfer and electron transfer steps, and determined their rates using stopped-flow methods. Hydride exchange between the enzyme and NADP(H) occurred at 30-150 s(-)(1), indicating that this half-reaction does not limit FPR activity. In contrast, electron transfer to flavodoxin proceeds at 2.7 s(-)(1), in the range of steady-state catalysis. Flavodoxin semiquinone was a better electron acceptor for FPR than oxidized flavodoxin under both single turnover and steady-state conditions. The results indicate that one-electron reduction of oxidized flavodoxin limits the enzyme activity in vitro, and support the notion that flavodoxin oscillates between the semiquinone and fully reduced states when FPR operates in vivo.

Anabaena↗

PubMed2004

Towards a new interaction enzyme:coenzyme.

Ferredoxin-NADP(+) reductase catalyses NADP(+) reduction, being specific for NADP(+)/H. To understand coenzyme specificity determinants and coenzyme specificity reversion, mutations at the NADP(+)/H pyrophosphate binding and of the C-terminal regions have been simultaneously introduced in Anabaena FNR. The T155G/A160T/L263P/Y303S mutant was produced. The mutated enzyme presents similar k(cat) values for NADPH and NADH, around 2.5 times slower than that reported for WT FNR with NADPH. Its K(m) value for NADH decreased 20-fold with regard to WT FNR, whereas the K(m) for NADPH remains similar. The combined effect is a much higher catalytic efficiency for NAD(+)/H, with a minor decrease of that for NADP(+)/H. In the mutated enzyme, the specificity for NADPH versus NADH has been decreased from 67,500 times to only 12 times, being unable to discriminate between both coenzymes. Additionally, giving the role stated for the C-terminal Tyr in FNR, its role in the energetics of the FAD binding has been analysed.

Anabaena↗

PubMed2004

Role of the C-terminal tyrosine of ferredoxin-nicotinamide adenine dinucleotide phosphate reductase in the electron transfer processes with its protein partners ferredoxin and flavodoxin.

The catalytic mechanism proposed for ferredoxin-NADP(+) reductase (FNR) is initiated by reduction of its flavin adenine dinucleotide (FAD) cofactor by the obligatory one-electron carriers ferredoxin (Fd) or flavodoxin (Fld) in the presence of oxidized nicotinamide adenine dinucleotide phosphate (NADP(+)). The C-terminal tyrosine of FNR, which stacks onto its flavin ring, modulates the enzyme affinity for NADP(+)/H, being removed from this stacking position during turnover to allow productive docking of the nicotinamide and hydride transfer. Due to its location at the substrate-binding site, this residue might also affect electron transfer between FNR and its protein partners. We therefore studied the interactions and electron-transfer properties of FNR proteins mutated at their C-termini. The results obtained with the homologous reductases from pea and Anabaena PCC7119 indicate that interactions with Fd or Fld are hardly affected by replacement of this tyrosine by tryptophan, phenylalanine, or serine. In contrast, electron exchange is impaired in all mutants, especially in the nonconservative substitutions, without major differences between the eukaryotic and the bacterial FNR. Introduction of a serine residue shifts the flavin reduction potential to less negative values, whereas semiquinone stabilization is severely hampered, introducing further constraints to the one-electron-transfer processes. Thus, the C-terminal tyrosine of FNR plays distinct and complementary roles during the catalytic cycle, (i) by lowering the affinity for NADP(+)/H to levels compatible with steady-state turnover, (ii) by contributing to the flavin semiquinone stabilization required for electron splitting, and (iii) by modulating the rates of electron exchange with the protein partners.

Amino Acid Sequence↗

PubMed2002

Probing the role of glutamic acid 139 of Anabaena ferredoxin-NADP+ reductase in the interaction with substrates.

The role of the negative charge of the E139 side-chain of Anabaena Ferredoxin-NADP+ reductase (FNR) in steering appropriate docking with its substrates ferredoxin, flavodoxin and NADP+/H, that leads to efficient electron transfer (ET) is analysed by characterization of several E139 FNR mutants. Replacement of E139 affects the interaction with the different FNR substrates in very different ways. Thus, while E139 does not appear to be involved in the processes of binding and ET between FNR and NADP+/H, the nature and the conformation of the residue at position 139 of Anabaena FNR modulates the precise enzyme interaction with the protein carriers ferredoxin (Fd) and flavodoxin (Fld). Introduction of the shorter aspartic acid side-chain at position 139 produces an enzyme that interacts more weakly with both ET proteins. Moreover, the removal of the charge, as in the E139Q mutant, or the charge-reversal mutation, as in E139K FNR, apparently enhances additional interaction modes of the enzyme with Fd, and reduces the possible orientations with Fld to more productive and stronger ones. Hence, removal of the negative charge at position 139 of Anabaena FNR produces a deleterious effect in its ET reactions with Fd whereas it appears to enhance the ET processes with Fld. Significantly, a large structural variation is observed for the E139 side-chain conformer in different FNR structures, including the E139K mutant. In this case, a positive potential region replaces a negative one in the wild-type enzyme. Our observations further confirm the contribution of both attractive and repulsive interactions in achieving the optimal orientation for efficient ET between FNR and its protein carriers.

Anabaena↗