Characterization of a novel ferredoxin with N-terminal extension from Clostridium acetobutylicum ATCC 824
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O RI G I NAL PAPE R
Characterization of a novel ferredoxin with N-terminal extension from Clostridium acetobutylicum ATCC 824 Razia Kutty · George N. Bennett
Received: 18 August 2006 / Revised: 27 September 2006 / Accepted: 4 October 2006 / Published online: 7 November 2006 © Springer-Verlag 2006
Abstract A gene (CAC2657) encoding a ferredoxin (EFR1) from the strictly anaerobic soil bacterium Clostridium acetobutylicum was cloned and expressed in Escherichia coli. The ferredoxin gene encodes a polypeptide of 27 kDa that incorporates 2[4Fe–4S] clusters. An extended N-terminal region of 187 amino acid (aa) residues precedes ferredoxin domain. The EFR1 expressed in E. coli is a trimeric protein. The iron and sulfur content of the reconstituted protein agrees with that expected of a trimeric form of the protein. The ferredoxin domain of EFR1 is closely related to ferredoxin of C. pasteurianum; and can be Wtted to the X-ray crystal structure with a root mean square deviation of 0.62 As for the C atoms of the generated 3D simulation model. In cultures of C. acetobutylicum the efr1 gene shows higher relative expression on induction with Trinitrotoluene (TNT) compared to that from uninduced control cultures. Keywords Ferredoxin · Polyferredoxin · Clostridium acetobutylicum · Electron acceptors · Trinitrotoluene biotransformation · Hydrogenase · Iron storage proteins · Iron–sulfur clusters
Introduction Ferredoxins are acidic low molecular weight proteins containing a pair of nonheme iron and acid-labile sulfur clusters coordinated by cysteines. These proteins R. Kutty · G. N. Bennett (&) Department of Biochemistry and Cell Biology MS-140, Rice University, Houston, TX 77005-1892, USA e-mail: [email protected]
participate in various redox reactions, and many organisms including Rhodospirillum rubrum (Yoch et al.1977), Streptomyces griseolus (O Keefe et al. 1991), and Clostridium thermoaceticum (Elliot and Ljungdahl 1982), contain ferredoxins. During the evolution of bacterial type ferredoxins intrasequence gene duplication, transposition and fusion events occurred, resulting in the appearance of proteins with multiple iron–sulphur centres. Polyferredoxins are proteins with multiple iron–sulfur centers containing tandemly repeated bacterial ferredoxin like domains. In Methanobacterium thermoautotrophicum strain H the gene mvhB encoding the 45 kDa polyferredoxin with 12[4Fe–4S] has been described, MvhB is a part of the mvhDGAB operon encoding methylviologen-reducing hydrogenase, an enzyme involved in methanogenesis from H2 and CO2 (Reeve et al. 1989; Weiss and Thauer 1993; Steigerwald et al. 1992). In Methanosarcina barkeri a polyferredoxin forms a tight complex with formylmethanofuran dehydrogenase (Vorholt et al. 1996). The association of polyferredoxin with hydrogenases in both the reports indicates a possible involvement in the reactions of hydrogenase. The functional role of polyferredoxins are not well documented (Reeve et al. 1989; Hedderich et al. 1992). The mvhB gene product has been proposed to function in associ
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