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 ∼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 ∼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.
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology