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

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.

Metal/protein ratio determination in the Rhodobacter capsulatus cytoplasmic pyrophosphatase enzyme by particle induced X-ray emission.

Inorganic pyrophosphatases are divided in two families, which differ both in structure and mechanism. All of them incorporate in its structure divalent metal cations. In 2003, it was reported for the first time that Rhodobacter capsulatus cytoplasmic pyrophosphatase belongs to family II. It is expected then, that this enzyme contains metal elements in its structure; however, this characterization has not been carried out yet. A fine application of accelerators is the use of proton beams to induce X-ray emission (PIXE) for analyzing the composition of biological macromolecules. The purpose of this work is to complement R. capsulatus cytoplasmic pyrophosphatase characterization by determining the presence of metal elements in its structure. Three different strategies were used: PAGE-PIXE, PAGE-Digestion-PIXE, and Dialysis-PIXE and when metals were found the metal/enzyme ratio was calculated. Only cobalt was found to be associated to the enzyme chemical structure in a ratio 3 Co/enzyme.

Coenzyme binding and hydride transfer in Rhodobacter capsulatus ferredoxin/flavodoxin NADP(H) oxidoreductase.

Ferredoxin-NADP(H) reductases catalyse the reversible hydride/electron exchange between NADP(H) and ferredoxin/flavodoxin, comprising a structurally defined family of flavoenzymes with two distinct subclasses. Those present in Gram-negative bacteria (FPRs) display turnover numbers of 1-5 s(-1) while the homologues of cyanobacteria and plants (FNRs) developed a 100-fold activity increase. We investigated nucleotide interactions and hydride transfer in Rhodobacter capsulatus FPR comparing them to those reported for FNRs. NADP(H) binding proceeds as in FNRs with stacking of the nicotinamide on the flavin, which resulted in formation of charge-transfer complexes prior to hydride exchange. The affinity of FPR for both NADP(H) and 2'-P-AMP was 100-fold lower than that of FNRs. The crystal structure of FPR in complex with 2'-P-AMP and NADP(+) allowed modelling of the adenosine ring system bound to the protein, whereas the nicotinamide portion was either not visible or protruding toward solvent in different obtained crystals. Stabilising contacts with the active site residues are different in the two reductase classes. We conclude that evolution to higher activities in FNRs was partially favoured by modification of NADP(H) binding in the initial complexes through changes in the active site residues involved in stabilisation of the adenosine portion of the nucleotide and in the mobile C-terminus of FPR.

The oxidant-responsive diaphorase of Rhodobacter capsulatus is a ferredoxin (flavodoxin)-NADP(H) reductase.

Challenge of Rhodobacter capsulatus cells with the superoxide propagator methyl viologen resulted in the induction of a diaphorase activity identified as a member of the ferredoxin (flavodoxin)-(reduced) nicotinamide adenine dinucleotide phosphate (NADP(H)) reductase (FPR) family by N-terminal sequencing. The gene coding for Rhodobacter FPR was cloned and expressed in Escherichia coli. Both native and recombinant forms of the enzyme were purified to homogeneity rendering monomeric products of approximately 30 kDa with essentially the same spectroscopic and kinetic properties. They were able to bind and reduce Rhodobacter flavodoxin (NifF) and to mediate typical FPR activities such as the NADPH-driven diaphorase and cytochrome c reductase.

The single superoxide dismutase of Rhodobacter capsulatus is a cambialistic, manganese-containing enzyme.

The phototrophic bacterium Rhodobacter capsulatus contains a single, oxygen-responsive superoxide dismutase (SOD(Rc)) homologous to iron-containing superoxide dismutase enzymes. Recombinant SOD(Rc), however, displayed higher activity after refolding with Mn(2+), especially when the pH of the assay mixture was raised. SOD(Rc) isolated from Rhodobacter cells also preferentially contains manganese, but metal discrimination depends on the culture conditions, with iron fractions increasing from 7% in aerobic cultures up to 40% in photosynthetic cultures. Therefore, SOD(Rc) behaves as a Mn-containing dismutase with cambialistic properties.

Molecular cloning and expression analysis of the Rhodobacter capsulatus sodB gene, encoding an iron superoxide dismutase.

Genetic complementation of a sodA sodB Escherichia coli mutant strain was used to clone Rhodobacter capsulatus genes involved in detoxification of superoxide radicals. After sequence analysis, 1 of the 16 identical clones obtained by this selection procedure was shown to contain an open reading frame with sequence similarity to that coding for Fe-containing superoxide dismutases (SodB). The R. capsulatus sodB gene was expressed in E. coli, and the nature of the metal ligand was confirmed by inhibitor sensitivity assays with lysates from both bacterial species. Activity staining of cleared Rhodobacter lysates resolved by polyacrylamide gel electrophoresis indicated that SodB was the only superoxide dismutase present in this phototrophic organism. The sodB gene was expressed at low levels in R. capsulatus cells grown under anaerobic or semiaerobic conditions, but expression was strongly induced upon exposure of the bacteria to air or to methyl viologen. Attempts to construct a sodB mutant in this organism by allelic exchange of the chromosomal copy of the gene with a suicide plasmid containing a mutated sodB gene were unsuccessful, strongly suggesting that the encoded superoxide dismutase is essential for viability of R. capsulatus in aerobic cultures.