title: A Rhizobium meliloti ferredoxin (FdxN) purified from Escherichia coli donates electrons to Rhodobacter capsulatus nitrogenase.
abstract: 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.
authors.affiliation: Lehrstuhl für Genetik, Fakultät für Biologie, Universität Bielefeld, Germany.
authors.forename: K U
authors.initials: KU
authors.lastname: Riedel
authors.forename: Y
authors.initials: Y
authors.lastname: Jouanneau
authors.forename: B
authors.initials: B
authors.lastname: Masepohl
authors.forename: A
authors.initials: A
authors.lastname: Pühler
authors.forename: W
authors.initials: W
authors.lastname: Klipp
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D004926:Escherichia coli / Q000235:genetics*; D005288:Ferredoxins / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D009591:Nitrogenase / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D016354:Rhodobacter capsulatus / Q000201:enzymology*; D016962:Sinorhizobium meliloti / Q000737:chemistry* / Q000235:genetics
chemical_list: D005288:Ferredoxins; D011994:Recombinant Proteins; D009591:Nitrogenase