CHARACTERIZATION OF AN IRON-SULFUR FLAVOPROTEIN FROM METHANOSARCINA-THERMOPHILA

A gene (isf) encoding an iron sulfur flavoprotein (Isf) from Methanosa rcina thermophila was cloned and sequenced. The gene was located direc tly upstream of the genes (pta and ach) encoding phosphotransacetylase and acetate kinase and is transcribed in the opposite direction. The amino acid sequence deduced from isf contained a cluster of cysteine r esidues reminiscent of proteins that accommodate either a [4Fe-4S] or [3Fe-4S] center. The protein was heterologously produced in Escherichi a coli and purified to apparent homogeneity. The 29-kDa subunit molecu lar mass of heterologously produced Isf (determined by SDS-polyacrylam ide gel electrophoresis) corresponded to the molecular mass of 30,451 Da calculated from the amino acid composition deduced from isf. Gel fi ltration estimated a molecular mass of 65 kDa for the native Isf-indic ating an alpha(2) homodimer. The UV-visible absorption spectrum was ch aracteristic 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 alpha(2) homodimer. Comparisons of the deduced Isf sequence with sequences in available protein data b ases suggested Isf is a novel iron-sulfur flavoprotein. Western blot a nalysis indicated the presence of Isf in extracts of acetate-grown M. thermophila. Ferredoxin stimulated the GO-dependant reduction of Isf b y the CO dehydrogenase-acetyl-CoA synthase complex that suggested ferr edoxin is a physiological electron donor to Isf.