The C-terminal extension of bacterial flavodoxin-reductases: Involvement in the hydride transfer mechanism from the coenzyme

Abstract

To study the role of the mobile C-terminal extension present in bacterial class of plant type NADP(H):ferredoxin reductases during catalysis, we generated a series of mutants of the Rhodobacter capsulatus enzyme (RcFPR). Deletion of the six C-terminal amino acids beyond alanine 266 was combined with the replacement A266Y, emulating the structure present in plastidic versions of this flavoenzyme. Analysis of absorbance and fluorescence spectra suggests that deletion does not modify the general geometry of FAD itself, but increases exposure of the flavin to the solvent, prevents a productive geometry of FAD:NADP(H) complex and decreases the protein thermal stability. Although the replacement A266Y partially coats the isoalloxazine from solvent and slightly restores protein stability, this single change does not allow formation of active charge-transfer complexes commonly present in the wild-type FPR, probably due to restraints of C-terminus pliability. A proton exchange process is deduced from ITC measurements during coenzyme binding. All studied RcFPR variants display higher affinity for NADP(+) than wild-type, evidencing the contribution of the C-terminus in tempering a non-productive strong (rigid) interaction with the coenzyme. The decreased catalytic rate parameters confirm that the hydride transfer from NADPH to the flavin ring is considerably hampered in the mutants. Although the involvement of the C-terminal extension from bacterial FPRs in stabilizing overall folding and bent-FAD geometry has been stated, the most relevant contributions to catalysis are modulation of coenzyme entrance and affinity, promotion of the optimal geometry of an active complex and supply of a proton acceptor acting during coenzyme binding.

Enhancement of Photophysical and Photosensitizing Properties of Flavin Adenine Dinucleotide by Mutagenesis of the C‐Terminal Extension of a Bacterial Flavodoxin Reductase

“…Some general steady‐state spectroscopic features of the Rc FPR variants indicated in Figure 1 were recently presented by Bortolotti et al2b In this work, we studied in more detail both spectroscopic and photophysical features of the Rc FPR series. Figure 2 shows a comparison of the UV/Vis absorption spectra of free FAD with both WT and A266YΔ enzymes in Tris buffer (50 m M pH 8).…”

“…The impact on the catalytic efficiency of both types of reductases by changes on the amino acid sequence and architecture of the C‐terminal region belonging to the NADP(H)‐binding domain, has been reviewed 1. 2 In a recent report, the role of the mobile C‐terminal extension during catalysis of the FPR of Rhodobacter capsulatus ( Rc FPR) was analyzed by deletion of the hexapeptide beyond Ala266 (Δ=FVGEGI) and in combination with the replacement of Ala266 by Tyr, emulating the structure present in plastidic versions of these flavoenzymes 2b. Although the changes in the C‐terminal domain did not significantly alter the global structure of the protein, they promoted the thermal instability of the mutants and also prevented the optimal geometry of the active FAD–NADP(H) charge‐transfer (CT) complex being achieved.…”

“…Furthermore, all Rc FPR variants displayed higher affinity for NADP + than the WT, evidence of the contribution of the C terminus in tempering a non‐productive strong (rigid) interaction with the coenzyme 2. In addition, preliminary absorbance and fluorescence spectral analysis of FAD for the Rc FPR mutants suggested that the C‐terminal modification does not greatly change the general geometry of the FAD binding site, but increases the exposure of the flavin isoalloxazine ring to the solvent 2b…”

ArabidopsisFNRL protein is an NADPH‐dependent chloroplast oxidoreductase resembling bacterial ferredoxin‐NADP+reductases

“…FNRL is considerably slower in the HT process from NADPH than the leaf‐type, root‐type or bacterial FNRs (Fig. ; Tejero et al , Bortolotti et al , Sánchez‐Azqueta et al , ). Moreover, the reduction of FNRL by NADPH differs with respect to the stabilisation of FNRL CTCs (Tejero et al , Peregrina et al , Sánchez‐Azqueta et al , , Bortolotti et al ).…”

“…Moreover, the reduction of FNRL by NADPH differs with respect to the stabilisation of FNRL CTCs (Tejero et al , Peregrina et al , Sánchez‐Azqueta et al , , Bortolotti et al ). Notably, differences in CTCs stabilisation have also been reported between bacterial‐type FPRs relative to plastidic FNRs, as well as upon mutation of key residues in plastid FNRs (Lans et al , Peregrina et al , Bortolotti et al , Sánchez‐Azqueta et al ). Such differences are related to dissimilarities in the geometric disposition of the reacting rings in the catalytically competent complexes, in the nature of residues at the re ‐face of the isoalloxazine where the coenzyme nicotinamide moiety is expected to stack for catalysis, or to the bipartite binding of the coenzyme (Sánchez‐Azqueta et al ).…”