Ferredoxin-like Protein Research Articles

The Bradyrhizobium japonicum fixBCX operon: identification of fixX and of a 5’mRNA region affecting the level of the fixSCX transcript

The Bradyrhizobium japonicum fixX gene was identified and shown to be essential for symbiotic and free-living, microaerobic nitrogen fixation. The fixX gene encodes a ferredoxin-like protein which may be involved in a redox process (electron transport?) essential for nitrogenase activity. This gene was localized downstream of fixC and its expression was dependent on the fixB promoter, providing evidence for the existence of a fixBCX operon. Mutagenesis and sequence analysis of the unusually long, 709bp leader region between the fixB promoter and the fixB structural gene did not reveal the presence of a nif or fix gene that was absolutely essential for nitrogen fixation. However, a short open reading frame (ORF) within this region encoding a polypeptide of 35 amino acids (ORF35) was shown to be efficiently translated. Chromosomal deletion of a 400bp DNA fragment covering ORF35 resulted in a three-fold reduction of the fixBCX mRNA level, which in turn also reduced the nitrogen fixation activity of this mutant. This suggests a possible post-transcriptional control mechanism for the expression of the fixBCX operon involving the stabilization of fixBCX mRNA by ribosomes actively translating ORF35.

A gene upstream of the Rhizobium trifolii nifA gene encodes a ferredoxin-like protein.

A gene upstream of the Rhizobium trifolii nifA gene encodes a ferredoxin-like protein.

Second gene (nif H* ) coding for a nitrogenase iron protein in Azotobacter chroococcum is adjacent to a gene coding for a ferredoxin-like protein

Azotobacter chroococcum MCD1 contains a cluster of nitrogen fixation (nif) genes coding for the structural polypeptides for nitrogenase (nifH for the Fe-protein and nifD and nifK for the MoFe protein) and a second sequence in the genome homologous to nifH. DNA fragments bearing this second nifH-like sequence were cloned and the DNA sequence around the homologous region determined. Two open reading frames were identified in this region. One codes for a protein of 289 amino acid residues and is highly homologous to other Fe-proteins but is different from the gene adjacent to the nifDK genes in A. chroococcum. This putative gene we call nifH*. The following open reading frame codes for a protein of 63 amino acids, nine of which are cysteine residues. The protein is homologous to the small low-potential ferredoxins found in anaerobic bacteria, and in particular those from Chlorobium limicola. Linkage between a structural gene for nitrogenase and a small ferredoxin has not previously been observed. Sequence analysis suggests that the two genes form an operon. Transcription of the ferredoxin gene on a 1320-bp transcript was only detectable under conditions in which A. chroococcum MCD1155, which carries a chromosomal deletion of 6.3 kb removing the entire nifHDK cluster, is capable of fixing N2, i.e. in media containing no added molybdenum or high levels of NH3. The size of the observed transcript agrees well with the predicted size for a transcript encoding nifH* and the ferredoxin genes. Expression of the nifH* promoter was not significantly activated in Escherichia coli even when nifA, the positive activator of nif genes in Klebsiella pneumoniae, was supplied in multiple copies. The results are discussed in relation to an alternative pathway for N2 fixation in A. chroococcum.

Iron-sulfur clusters in Azotobacter ferredoxin at 2.5 A resolution.

X-ray crystallographic study of the ferredoxin-like protein (iron-sulfur protein III) from Azotobacter vinelandii has been extended to 2.5-A resolution. A 4.0-A resolution electron density map revealed that the molecule contains two Fe-S clusters of differing size and shape separated by 12 A (Stout, C.D. (1979) Nature 279, 83-84). Recent Mössbauer results by Emptage et al. (Emptage, M.H., Kent, T.A., Huynh, B.H., Rawlings, J., Orme-Johnson, W.H., and Münck, E. (1980) J. Biol. Chem. 255, 1793-1796) have shown that the molecule contains 7 iron atoms as a high potential iron protein-like Fe4 center and a novel 3-Fe center. The 2.5-A electron density map shows two distinctly different Fe-S clusters. The larger cluster consists of a tetranuclear [4Fe-4S] core ligated to the protein at each iron atom. The smaller cluster is distinctly planar and cannot be modeled with [2Fe-2S] or [4Fe-4S] structures. The best model for this cluster is a [3Fe-3S] core. The protein makes six contacts with this cluster.

Structure of the iron–sulphur clusters in Azotobacter ferredoxin at 4.0 Å resolution

A FERREDOXIN-LIKE protein from Azotobacter vinelandii having 6–7 Fe and 6–7 S2− per mol and a molecular weight of 14,000 has been described by Shethna1 and Yoch et al.2. Its biochemical properties3, electron paramagnetic resonance and redox behaviour4,5, and primary sequence6 have also been studied. This work has established that there are two Fe–S centres separated by 0.744 V in reduction potential, one behaving like the [Fe4S4S4Cys] cluster in high-potential iron proteins, the other displaying novel characteristics. The distribution of cysteines in the N-terminal sequence is distinctly non-homologous with clostridial ferredoxins. Extrusion of the Fe–S cores by thiol displacement produces unique species, suggesting the presence of a new chromophore structure7. I report here crystallographic studies of the protein in a tetragonal crystal form which have led to an electron density map at 4.0 A resolution. This map reveals two Fe–S clusters of clearly different size and shape. It has not been previously shown that this Fe–S protein, or any other, actually contains clusters of differing structure.

Immunohistochemical demonstration of an adrenal ferredoxin-like iron-sulfur protein in rat hepatic mitochondria.

Immunohistochemical demonstration of an adrenal ferredoxin-like iron-sulfur protein in rat hepatic mitochondria.