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.
Département de Biologie Moléculaire et Structurale, Centre d'Etudes Nucléaires de Grenoble, France.
Y
Y
Jouanneau
C
C
Meyer
J
J
Gaillard
E
E
Forest
J
J
Gagnon
D000595:Amino Acid Sequence; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D017886:Durapatite; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D005288:Ferredoxins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006882:Hydroxyapatites; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009591:Nitrogenase / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D016354:Rhodobacter capsulatus / Q000378:metabolism*; D013053:Spectrophotometry
D005288:Ferredoxins; D006882:Hydroxyapatites; D046911:Macromolecular Substances; D017886:Durapatite; D009591:Nitrogenase