Catalysis in fumarate reductase

In the absence of oxygen many bacteria are able to utilise fumarate as a te rminal oxidant for respiration. In most known organisms the fumarate reduct ases are membrane-bound iron-sulfur flavoproteins but Shewanella species pr oduce a soluble, periplasmic flavocytochrome c(3) that catalyses this react ion. The active sites of all fumarate reductases are clearly conserved at t he structural level, indicating a common mechanism. The structures of fumar ate reductases from two Shewanella species have been determined. Fumarate, succinate and a partially hydrated fumarate ligand are found in equivalent locations in different crystals, tightly bound in the active site and close to N5 of the FAD cofactor, allowing identification of amino acid residues that are involved in substrate binding and catalysis. Conversion of fumarat e to succinate requires hydride transfer from FAD and protonation by an act ive site acid. The identity of the proton donor has been open to question b ut we have used structural considerations to suggest that this function is provided by an arginine side chain. We have confirmed this experimentally b y analysing the effects of site-directed mutations on enzyme activity. Subs titutions of Arg402 lead to a dramatic loss of activity whereas neither of the two active site histidine residues is required for catalysis. (C) 2000 Elsevier Science B.V. All rights reserved.