A POLYFERREDOXIN WITH 8 [4FE-4S] CLUSTERS AS A SUBUNIT OF MOLYBDENUM FORMYLMETHANOFURAN DEHYDROGENASE FROM METHANOSARCINA-BARKERI

Formylmethanofuran dehydrogenase (Fmd) from Methanosarcina barkeri is a molybdenum iron-sulfur protein involved in methanogenesis. The enzym e contains approximately 30 mel non-heme iron/mol and 30 mol acid-labi le sulfur/mol. We report here the cloning and sequencing of the encodi ng genes, and that these genes form a transcription unit findEFACDB. E vidence is provided that the subunit FmdB harbours the molybdenum-cont aining active site and may bind one [4Fe-4S] cluster. fmdF encodes a p rotein with four tandemly repeated bacterial-ferredoxin-like domains a nd is predicted to be a polyferredoxin that could contain as many as 3 2 iron atoms in eight [4Fe-4S] clusters. The other genes code for prot eins without sequence motifs characteristic for iron-sulfur proteins. These findings suggest that most of the iron-sulfur clusters present i n the purified formylmethanofuran dehydrogenase are associated with th e subunit FmdF The finding that FmdF forms a tight complex with the ot her subunits of formylmethanofuran dehydrogenase indicates a function of the polyferredoxin in the reaction catalyzed by the enzyme. fmdE en codes a protein not present in the purified enzyme. All six genes of t he find operon were expressed in Escherichia coli and yielded proteins of expected molecular masses. A malE-findF gene fusion was constructe d and expressed in E. coli, making the apoprotein of the polyferredoxi n available in preparative amounts.