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Volume 271, Number 39, Issue of September 27, 1996 pp. 24023-24028
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Characterization of an Iron-Sulfur Flavoprotein from Methanosarcina thermophila

(Received for publication, April 26, 1996, and in revised form, July 15, 1996)

From the Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802-4500 and the Department of Biochemistry and Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061-0305

A gene (isf) encoding an iron-sulfur flavoprotein (Isf) from Methanosarcina thermophila was cloned and sequenced. The gene was located directly upstream of the genes (pta and ack) encoding phosphotransacetylase and acetate kinase and is transcribed in the opposite direction. The amino acid sequence deduced from isf contained a cluster of cysteine residues reminiscent of proteins that accommodate either a [4Fe-4S] or [3Fe-4S] center. The protein was heterologously produced in Escherichia coli and purified to apparent homogeneity. The 29-kDa subunit molecular mass of heterologously produced Isf (determined by SDS-polyacrylamide gel electrophoresis) corresponded to the molecular mass of 30,451 Da calculated from the amino acid composition deduced from isf. Gel filtration estimated a molecular mass of 65 kDa for the native Isf indicating an ₂ homodimer. The UV-visible absorption spectrum was characteristic of iron-sulfur flavoproteins with maxima at 484, 452, 430, 378, and 280 nm. Analyses identified 2 FMN, 7-8 non-heme iron atoms, and 6-7 acid-labile sulfur atoms per ₂ homodimer. Comparisons of the deduced Isf sequence with sequences in available protein data bases suggested Isf is a novel iron-sulfur flavoprotein. Western blot analysis indicated the presence of Isf in extracts of acetate-grown M. thermophila. Ferredoxin stimulated the CO-dependant reduction of Isf by the CO dehydrogenase·acetyl-CoA synthase complex that suggested ferredoxin is a physiological electron donor to Isf.

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