title: Spectroscopic and functional properties of novel 2[4Fe-4S] cluster-containing ferredoxins from the green sulfur bacterium Chlorobium tepidum.
abstract: Two distinct ferredoxins, Fd I and Fd II, were isolated and purified to homogeneity from photoautotrophically grown Chlorobium tepidum, a moderately thermophilic green sulfur bacterium that assimilates carbon dioxide by the reductive tricarboxylic acid cycle. Both ferredoxins serve a crucial role as electron donors for reductive carboxylation, catalyzed by a key enzyme of this pathway, pyruvate synthase/pyruvate ferredoxin oxidoreductase. The reduction potentials of Fd I and Fd II were determined by cyclic voltammetry to be -514 and -584 mV, respectively, which are more electronegative than any previously studied Fds in which two [4Fe-4S] clusters display a single transition. Further spectroscopic studies indicated that the CD spectrum of oxidized Fd I closely resembled that of Fd II; however, both spectra appeared to be unique relative to ferredoxins studied previously. Double integration of the EPR signal of the two Fds yielded approximately approximately 2.0 spins per molecule, compatible with the idea that C. tepidum Fd I and Fd II accept 2 electrons upon reduction. These results suggest that the C. tepidum Fd I and Fd II polypeptides each contain two bound [4Fe-4S] clusters. C. tepidum Fd I and Fd II are novel 2[4Fe-4S] Fds, which were shown previously to function as biological electron donors or acceptors for C. tepidum pyruvate synthase/pyruvate ferredoxin oxidoreductase (Yoon, K.-S., Hille, R., Hemann, C. F., and Tabita, F. R. (1999) J. Biol. Chem. 274, 29772-29778). Kinetic measurements indicated that Fd I had approximately 2.3-fold higher affinity than Fd II. The results of amino acid sequence alignments, molecular modeling, oxidation-reduction potentials, and spectral properties strongly indicate that the C. tepidum Fds are chimeras of both clostridial-type and chromatium-type Fds, suggesting that the two Fds are likely intermediates in the evolutional development of 2[4Fe-4S] clusters compared with the well described clostridial and chromatium types.
authors.affiliation: Department of Microbiology, Plant Molecular Biology/Biotechnology Program, Protein Research Group, the Ohio State University, Columbus, Ohio 43210-1292, USA.
authors.forename: K S
authors.initials: KS
authors.lastname: Yoon
authors.forename: C
authors.initials: C
authors.lastname: Bobst
authors.forename: C F
authors.initials: CF
authors.lastname: Hemann
authors.forename: R
authors.initials: R
authors.lastname: Hille
authors.forename: F R
authors.initials: FR
authors.lastname: Tabita
mesh_terms: D000595:Amino Acid Sequence; D019414:Chlorobi / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D005288:Ferredoxins / Q000737:chemistry / Q000378:metabolism*; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid
chemical_list: D005288:Ferredoxins; D007506:Iron-Sulfur Proteins