A Rhizobium meliloti ferredoxin (FdxN) purified from Escherichia coli donates electrons to Rhodobacter capsulatus nitrogenase.

The fdxN gene from Rhizobium meliloti encoding a bacterial-type ferredoxin (FdxN) was expressed in Escherichia coli under the control of the lac promoter. The fdxN gene product was purified under anaerobic conditions by ion-exchange chromatography and gel-filtration steps using an antiserum raised against an FdxN-LacZ fusion protein as a detection system. The purified ferredoxin was shown to be identical to the predicted R. meliloti FdxN protein in its amino acid composition and N-terminal amino acid sequence. Chemical determination of the iron content revealed 8.6 +/- 0.6 mol Fe/mol FdxN. The ultraviolet/visible absorption spectrum of the FdxN protein in the oxidized form exhibited maxima at 284 nm and 378 nm, with an A378/A284 ratio of 0.7. EPR spectroscopy revealed a rhombic signal when FdxN was partially reduced, and a broad signal indicative of spin-spin interaction when fully reduced, suggesting the presence of two Fe-S cluster/ferredoxin polypeptide. Our data suggest that FdxN contains two [4Fe-4S] clusters. Purified FdxN was able to mediate electron transport between illuminated chloroplasts and Rhodobacter capsulatus nitrogenase in vitro.

Riedel KU ，Jouanneau Y ，Masepohl B ，Pühler A ，Klipp W ... -  《european journal of biochemistry》

Characterization of a 2[4Fe-4S] ferredoxin obtained by chemical insertion of the Fe-S clusters into the apoferredoxin II from Rhodobacter capsulatus.

The Rhodobacter capsulatus ferredoxin II (FdII) belongs to a family of 7Fe ferredoxins containing one [3Fe-4S] cluster and one [4Fe-4S] cluster. This protein, encoded by the fdxA gene, has been overproduced in Escherichia coli as a soluble apoferredoxin. The purified recombinant protein was subjected to reconstitution experiments by chemical incorporation of the Fe-S clusters under anaerobic conditions. A brown protein was obtained, the formation of which was dependent upon the complete unfolding of the polypeptide prior to incorporation of iron and sulfur atoms. The yield of the reconstituted product was higher when the reaction was carried out at slightly basic pH. The reconstituted ferredoxin was purified and shown to be distinct from the native [7Fe-8S] ferredoxin, based on several biochemical and spectroscopic criteria. In the oxidized state, EPR revealed the quasi-absence of [3Fe-4S] cluster. 1H-NMR spectroscopic analyses provided evidence that the protein was reconstituted as a 2[4Fe-4S] ferredoxin. This conclusion was further supported by the determination by electrospray mass spectrometry of the molecular mass of the reconstituted protein, which matched within 2 Da to the mass of the FdII polypeptide incremented of eight atoms each of iron and sulfur. Exposure of the reconstituted protein to air resulted in a fast and irreversible oxidative denaturation of the Fe-S clusters, without formation of [7Fe-8S] form. Unlike the natural 7Fe ferredoxin, the reconstituted ferredoxin appeared incompetent in an electron-transfer assay coupled to nitrogenase activity. The fact that the apoFdII was reconstituted as a highly unstable 8Fe ferredoxin instead of the 7Fe naturally occurring FdII is discussed in relation to the results obtained with other types of ferredoxins.

Armengaud J ，Gaillard J ，Forest E ，Jouanneau Y ... -  《european journal of biochemistry》

Characterization of an fdxN mutant of Rhodobacter capsulatus indicates that ferredoxin I serves as electron donor to nitrogenase.

A mutant of Rhodobacter capsulatus, carrying an insertion into the fdxN gene encoding ferredoxin I (FdI), has been studied by biochemical analysis and genetic complementation experiments. When compared to the wild-type strain, the fdxN mutant exhibited altered nitrogen fixing ability and 20-fold lower levels of nitrogenase activity as assayed in vivo. When assayed in vitro with an artificial reductant, nitrogenase activity was only 3- to 4-fold lower than in the wild type. These results suggested that the FdI-deleted mutant had impaired electron transport to nitrogenase. Immunochemical assay of both nitrogenase components showed that the fdxN mutant contained about 4-fold less enzyme than wild-type cells. Results of pulse-chase labeling experiments using [35S]methionine indicated that nitrogenase was significantly less stable in the FdI-deleted mutant. When a copy of fdxN was introduced in the mutant in trans, the resulting strain appeared to be fully complemented with respect to both diazotrophic growth and nitrogenase activity. Depending on whether fdxN expression was driven by a nif promoter or a fructose-inducible promoter, FdI was synthesized either at wild-type level or in 10-fold lower amounts. The strain producing 10-fold less FdI did, however, display normal N2-fixing ability. Analysis of cytosolic proteins by bidimensional electrophoresis revealed that the fdxN mutant produced a 14 kDa polypeptide in amounts about 3-fold greater than wild-type cells. This protein was identified by N-terminal microsequencing as a recently purified [2Fe-2S] ferredoxin, called FdV, which cannot reduce nitrogenase. It is concluded that FdI serves as the main electron donor to nitrogenase in R. capsulatus and that an ancillary electron carrier, distinct of FdV, is responsible for the residual nitrogenase activity observed in the FdI-deleted mutant.

Jouanneau Y ，Meyer C ，Naud I ，Klipp W ... -  《-》

Purification and characterization of a novel dimeric ferredoxin (FdIII) from Rhodobacter capsulatus.

A new ferredoxin, called FdIII, has been isolated and purified from the photosynthetic bacterium Rhodobacter capsulatus. Its complete amino acid sequence has been determined. The FdIII polypeptide consists of 100 residues, including 9 cysteines and has a calculated molecular mass of 10,688 Da, which was confirmed by electrospray mass spectrometry. In its native form, FdIII is a homodimer as deduced from molecular sieve chromatography and non-denaturing polyacrylamide gel electrophoresis, as well as cross-linking experiments. The dimeric ferredoxin was found to contain 15.2 +/- 0.6 iron atoms and 13 +/- 1 inorganic sulfur atoms, consistent with the presence of four [4Fe-4S] clusters/molecule. The UV visible absorption spectrum of oxidized FdIII exhibited maxima at 282 and at 386 nm and a shoulder near 314 nm. FdIII was fully reduced by excess dithionite at pH 8.0 or photochemically using 5-deazaflavin. Anaerobic oxidative titration of reduced FdIII with thionin indicated that each FdIII monomer exchanges two electrons. Exposure of FdIII to air resulted in a rapid and irreversible oxidative denaturation of the Fe-S clusters. The EPR spectrum of fully reduced FdIII showed a broad signal with an average g value of 1.94 that integrated to about two spins/monomer. EPR analysis of partially reduced FdIII (approximately 20% reduction) revealed a complex set of signals which was interpreted as being the resulting sum of the contribution of two distinct paramagnetic centres. Based on its biochemical and spectroscopic properties, it is concluded that FdIII is a dimeric ferredoxin containing four [4Fe-4S] clusters. The synthesis of FdIII occurs only under growth conditions allowing the derepression of nif genes. Results of in vitro electron transfer assays indicate that FdIII cannot serve as an electron donor to nitrogenase.

Jouanneau Y ，Meyer C ，Gaillard J ，Forest E ，Gagnon J ... -  《JOURNAL OF BIOLOGICAL CHEMISTRY》

A new [2Fe-2S] ferredoxin from Rhodobacter capsulatus. Coexpression with a 2[4Fe-4S] ferredoxin in Escherichia coli.

A 285-base pair open reading frame was found immediately upstream of the fdxN gene (encoding ferredoxin I) of Rhodobacter capsulatus and coded for a 95-amino acid protein with a predicted molecular weight of 10,156. The deduced amino acid sequence contained 5 cysteines, 4 of which exhibited spacing characteristic of [2Fe-2S] plant and cyanobacterial ferredoxins. The amino acid sequence was found to share approximately 25% amino acid similarity with plant-type ferredoxins. The gene was named fdxC. Expression of the fdxC and fdxN genes together in Escherichia coli was accomplished by subcloning the genes in the vector pUC18 downstream of the lac promoter. Cells containing this plasmid produced a red and a brown protein corresponding to the fdxC and fdxN gene products, respectively. EPR and UV-visible absorption spectroscopy confirmed that the FdxC protein contained a [2Fe-2S] cluster and the FdxN protein contained two [4Fe-4S] clusters and that the centers were correctly assembled and inserted in the ferredoxins expressed in E. coli. Transcription (Northern blot) analysis showed that the genes were transcribed only under nitrogen-limiting (nif-derepressing) growth conditions.

Grabau C ，Schatt E ，Jouanneau Y ，Vignais PM ... -  《JOURNAL OF BIOLOGICAL CHEMISTRY》