A Rhizobium Meliloti Ferredoxin (FdxN) Purified from Escherichia Coli Donates Electrons to Rhodobacter Capsulatus Nitrogenase
| Autor: | Alfred Pühler, Bernd Masepohl, Werner Klipp, Kai-Uwe Riedel, Yves Jouanneau |
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| Rok vydání: | 1995 |
| Předmět: |
Blotting
Western Molecular Sequence Data lac operon NITROGEN FIXATION medicine.disease_cause Biochemistry Rhodobacter capsulatus Electron Transport Escherichia Nitrogenase Escherichia coli medicine ELECTRON TRANSFER Amino Acid Sequence Peptide sequence Ferredoxin Rhodobacter biology Electron Spin Resonance Spectroscopy biology.organism_classification Recombinant Proteins Ferredoxins bacteria Rhizobium Sinorhizobium meliloti |
| Zdroj: | European Journal of Biochemistry. 231:742-746 |
| ISSN: | 1432-1033 0014-2956 |
| DOI: | 10.1111/j.1432-1033.1995.0742d.x |
| Popis: | The fdxN gene from Rhizobium meliloti encoding a bacterial-type ferredoxin (FdxN) was expressed in Escherichia coil 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 A(378)/A(284) 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 clusters/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. |
| Databáze: | OpenAIRE |
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