Mapping the interaction of the [2Fe-2S] Clostridium pasteurianum ferredoxin with nitrogenase MoFe protein.

The [2Fe-2S] ferredoxin from Clostridium pasteurianum had previously been shown to interact specifically with the nitrogenase MoFe protein, and electrostatic forces were found to be important contributors to the interaction. This phenomenon has now been analyzed in detail by using ferredoxin variants in which charge inversions or cancellations were introduced on all charged residues. The mutated forms of the ferredoxin were covalently cross-linked to the MoFe protein. The reaction products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and their nitrogenase activity was measured. The latter displayed a consistent inverse correlation with the amount of cross-linked MoFe protein. The data allowed an unambiguous identification of the ferredoxin residues (glutamates 31, 34, 38, 39, 84, 85) that are involved in the interaction with the MoFe protein. Furthermore, whereas the wild-type ferredoxin yielded approximately equal amounts of cross-linked products with the alpha and beta subunits of the MoFe protein, some of its molecular variants displayed a differential decrease of reactivity towards one or the other of these subunits. The positions on the ferredoxin molecule of the residues interacting with the MoFe protein were determined using the recently elucidated crystal structure of the homologous [2Fe-2S] ferredoxin from Aquifex aeolicus.

Carrageenan oligosaccharides enhance stress-induced microspore embryogenesis in Brassica oleracea var. italica.

Embryogenic induction in cultures of isolated microspores is a stress-dependent process, which can be triggered by heat shock, sucrose or nitrogen starvation or by anti-microtubular drugs. As they are known to mimic biotic stress, oligosaccharides were tested as an alternative source of compounds to induce microspore embryogenesis in Brassica oleracea var. italica. Among the eight oligosaccharide series that were investigated and the corresponding polysaccharides, namely pectin, alginate, fucan, laminarin, agar and kappa-, iota-, and lambda-carrageenans, only the carrageenan oligomers displayed significant effects on microspore induction. When supplied in combination to heat stress, they markedly enhanced the final yields of microspore-derived embryos, with a two-fold increase in the most reactive treatment, i.e. in the presence of lambda-carrageenan oligosaccharides. A 30 min treatment was enough to stimulate embryogenesis, and two optimal concentrations were observed, 170 nM and 34 &mgr;M.

Influence of the degree of acetylation on some biological properties of chitosan films.

In this study, we investigated in vitro the role of the degree of acetylation (DA) on some biological properties of chitosan films. We noticed that, whatever the DA, all chitosan films were cytocompatible towards keratinocytes and fibroblasts. We also demonstrated that the higher the DA of chitosan, the lower was the cell adhesion on the films. Fibroblasts appear to adhere twice as much as keratinocytes on these materials. We observed that keratinocyte proliferation increases when the DA of chitosan films decreases. Thus, DA influences the cell growth in the same way as cell adhesion. On the other hand, although they remain alive, fibroblasts do not proliferate on chitosan films. This behaviour is related to an extremely high adhesion on this kind of material, which certainly inhibits cell growth. In conclusion, DA plays a key role in cell adhesion and proliferation, but does not change the cytocompatibility of chitosan. In parallel, it is also important to notice the role played by the surface morphology of the material, a second major parameter which influences the mechanism of adhesion.

Structure of a thioredoxin-like [2Fe-2S] ferredoxin from Aquifex aeolicus.

The 2.3 A resolution crystal structure of a [2Fe-2S] cluster containing ferredoxin from Aquifex aeolicus reveals a thioredoxin-like fold that is novel among iron-sulfur proteins. The [2Fe-2S] cluster is located near the surface of the protein, at a site corresponding to that of the active-site disulfide bridge in thioredoxin. The four cysteine ligands are located near the ends of two surface loops. Two of these ligands can be substituted by non-native cysteine residues introduced throughout a stretch of the polypeptide chain that forms a protruding loop extending away from the cluster. The presence of homologs of this ferredoxin as components of more complex anaerobic and aerobic electron transfer systems indicates that this is a versatile fold for biological redox processes.

The [2Fe-2S] protein I (Shetna protein I) from Azotobacter vinelandii is homologous to the [2Fe-2S] ferredoxin from Clostridium pasteurianum.

The [2Fe-2S] protein from Azotobacter vinelandii that was previously known as iron-sulfur protein I, or Shethna protein I, has been shown to be encoded by a gene belonging to the major nif gene cluster. Overexpression of this gene in Escherichia coli yielded a dimeric protein of which each subunit comprises 106 residues and contains one [2Fe-2S] cluster. The sequence of this protein is very similar to that of the [2Fe-2S] ferredoxin from Clostridium pasteurianum (2FeCpFd), and the four cysteine ligands of the [2Fe-2S] cluster occur in the same positions. The A. vinelandii protein differs from the C. pasteurianum one by the absence of the N-terminal methionine, the presence of a five-residue C-terminal extension, and a lesser number of acidic and polar residues. The UV-visible absorption and EPR spectra, as well as the redox potentials of the two proteins, are nearly identical. These data show that the A. vinelandii FeS protein I, which is therefore proposed to be designated 2FeAvFdI, is the counterpart of the [2Fe-2S] ferredoxin from C. pasteurianum. The occurrence of the 2FeAvFdI-encoding gene in the nif gene cluster, together with the previous demonstration of a specific interaction between the 2FeCpFd and the nitrogenase MoFe protein, suggest that both proteins might be involved in nitrogen fixation, with possibly similar roles.

A [2Fe-2S] protein from the hyperthermophilic bacterium Aquifex aeolicus.

Overexpression in Escherichia coli of the fdx4 gene from Aquifex aeolicus has allowed isolation and characterization of the first hyperthermophilic [2Fe-2S](Scys)(4) protein, a homodimer of M = 2 x 12.4 kDa with one [2Fe-2S] cluster per subunit. This protein is undamaged by heating to 100 degrees C for at least three hours. The primary structure, in particular the characteristic distribution of the four cysteine ligands of the metal site, and the spectroscopic properties of the A. aeolicus protein relate it to well characterized [2Fe-2S] proteins from Clostridium pasteurianum and Azotobacter vinelandii. These proteins are also homologous to subunits or domains of hydrogenases and NADH-ubiquinone oxidoreductase (Complex I) of respiratory chains. The A. aeolicus [2Fe-2S] protein is thus representative of a presumably novel protein fold involved in a variety of functions in very diverse cellular backgrounds.

Extensive ligand rearrangements around the [2Fe-2S] cluster of Clostridium pasteurianum ferredoxin.

The [2Fe-2S] cluster of the ferredoxin from Clostridium pasteurianum is coordinated by cysteines 11, 56, and 60 and by a fourth cysteine, residue 24 in the wild-type protein, located on a flexible and deletable loop around residues 14-30. New mutated forms of this ferredoxin show that the fourth cysteine ligand can be located in any one of positions 14, 16, 21, 24, or 26. Another set of molecular variants has unveiled a new case of ligand swapping on the cysteine 60 ligand site. Replacement of cysteine 60 by alanine and introduction of a cysteine in position 21 yielded a ferredoxin that assembles a [2Fe-2S] cluster of which the ligands are cysteines 11, 21, 24, and 56. This cysteine ligand pattern is similar to that occurring in plant-type or mammalian-type ferredoxins, although the overall sequence similarities are below detection. Moreover, the vibrational and electronic properties of the resulting [2Fe-2S]2+/+ center, as revealed by resonance Raman and EPR studies, are strikingly similar to those of mammalian-type ferredoxins. The extensive set of mutated forms of the C. pasteurianum ferredoxin now available indicates that cysteine ligand exchange may occur on residues 24 and 60, but not on residues 11 and 56. It is thus suggested that cysteines 24 and 60 are part of a solvent accessible aspect of the Fe-S cluster, whereas cysteines 11 and 56 are buried and form the more rigid part of the polypeptide ligand framework. In view of the unprecedented versatility of this [2Fe-2S] cluster and of its polypeptidic environment, the introduction of ligands other than cysteine in various positions has been attempted. These experiments have remained unsuccessful, and even including previous studies, noncysteinyl ligation has been obtained with this protein in only very few cases. The data provide an extensive confirmation that Fe-S clusters have a strong preference for thiolate ligation and rationalize the relatively rare occurrence of noncysteinyl ligation in native Fe-S proteins.