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

Ki-Seok Yoon,F Robert Tabita,Cedric Bobst,Russ Hille,Craig F Hemann

doi:10.1074/jbc.m107852200

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.

Highlights

The reductive tricarboxylic acid (RTCA)1 cycle serves as an important pathway to enable diverse organisms to use CO2 as the sole source of carbon for growth
A purification scheme was devised for Fd I and Fd II consisting of sequential steps of DEAE-Sepharose fast flow, Q-Sepharose high performance, and Superose-12 chromatography, all performed in a Coy chamber under strictly anaerobic conditions at room temperature
C. tepidum Fd I and Fd II have typical two [4Fe-4S]2ϩ/1ϩ clusters that are coordinated in the first cluster via a CXXCXXCXXXCP motif and in the second cluster by a CXXCXXXXXXXXCXXXCP sequence motif

Summary

EXPERIMENTAL PROCEDURES

Bacterial Strains and Growth Conditions—C. tepidum strain TLS was grown photoautotrophically in 20-liter Carboy bottles according to a modification of a method described previously [3]. Protein purification was performed in the Coy anaerobic chamber using the Biological Autoprogram System from Bio-Rad. The supernatant after ultracentrifugation was loaded onto a DEAE-Sepharose fast flow column (2.5 ϫ 20 cm), preequilibrated with 20 mM Tris-HCl, pH 7.8, 10 mM ␤-mercaptoethanol. The column was washed with 150 ml of the same buffer containing 0.2 M NaCl at a flow rate of 7 ml per min, followed by elution with 600 ml of a linear gradient of 0.2-0.5 M NaCl prepared in the same buffer. The column was washed with buffer and protein eluted using buffer containing 0.3 M NaCl followed by a gradient of buffer containing 0.3– 0.5 M NaCl at a flow rate of 3 ml per min. 50 mM Tris-HCl, pH 8.0, containing either 0.1 or 1.0 M NaCl, 10 mM ␤-mercaptoethanol was used as the eluate buffer

RESULTS

Fd I Fd II MNZ

DISCUSSION

may be more accessible to oxygen molecules than those of