Spectroscopic and functional properties of novel 2[4Fe-4S] cluster-containing ferredoxins from the green sulfur bacterium Chlorobium tepidum

Two distinct ferredoxins, Fd I and Fd II, were isolated and purified to hom ogeneity from photoautotrophically grown Chlorobium tepidum, a moderately t hermophilic green sulfur bacterium that assimilates carbon dioxide by the r eductive 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 red uction potentials of Fd I and Fd II were determined by cyclic voltammetry t o be -514 and -584 mV, respectively, which are more electronegative than an y previously studied Fds in which two [4Fe-4S] clusters display a single tr ansition. Further spectroscopic studies indicated that the CD spectrum of o xidized Fd I closely resembled that of Fd Il; however, both spectra appeare d to be unique relative to ferredoxins studied previously. Double integrati on of the EPR signal of the two Fds yielded approximately similar to2.0 spi ns per molecule, compatible with the idea that C. tepidum Fd I and Fd II ac cept 2 electrons upon reduction. These results suggest that the C. tepidum Fd I and Fd Il polypeptides each contain two bound [4Fe-4S] clusters. C. te pidum Fd I and Fd II are novel 2[4Fe-4S] Fds, which were shown previously t o function as biological electron donors or acceptors for C. tepidum pyruva te synthase/pyruvate ferredoxin oxidoreductase (Yoon, K.-S., Hille, R., Hem ann, C. F., and Tabita, F. R. (1999) J. Biol. Chem. 274, 29772-29778). Kine tic measurements indicated that Fd I had similar to2.3-fold higher affinity than Fd IL The results of amino acid sequence alignments, molecular modeli ng, oxidation-reduction potentials, and spectral properties strongly indica te that the C. tepidum Fds are chimeras of both clostridial-type and chroma tium-type Fds, suggesting that the two Fds are likely intermediates in the evolutional development of 2[4Fe-4S] clusters compared with the well descri bed clostridial and chromatium types.