title: A proton delivery pathway in the soluble fumarate reductase from Shewanella frigidimarina.
abstract: The mechanism for fumarate reduction by the soluble fumarate reductase from Shewanella frigidimarina involves hydride transfer from FAD and proton transfer from the active-site acid, Arg-402. It has been proposed that Arg-402 forms part of a proton transfer pathway that also involves Glu-378 and Arg-381 but, unusually, does not involve any bound water molecules. To gain further insight into the importance of this proton pathway we have perturbed it by substituting Arg-381 by lysine and methionine and Glu-378 by aspartate. Although all the mutant enzymes retain measurable activities, there are orders-of-magnitude decreases in their k(cat) values compared with the wild-type enzyme. Solvent kinetic isotope effects show that proton transfer is rate-limiting in the wild-type and mutant enzymes. Proton inventories indicate that the proton pathway involves multiple exchangeable groups. Fast scan protein-film voltammetric studies on wild-type and R381K enzymes show that the proton transfer pathway delivers one proton per catalytic cycle and is not required for transporting the other proton, which transfers as a hydride from the reduced, protonated FAD. The crystal structures of E378D and R381M mutant enzymes have been determined to 1.7 and 2.1 A resolution, respectively. They allow an examination of the structural changes that disturb proton transport. Taken together, the results indicate that Arg-381, Glu-378, and Arg-402 form a proton pathway that is completely conserved throughout the fumarate reductase/succinate dehydrogenase family of enzymes.
authors.affiliation: Edinburgh and St. Andrews Research School of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ, UK.
authors.forename: Katherine L
authors.initials: KL
authors.lastname: Pankhurst
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Mowat
authors.forename: Emma L
authors.initials: EL
authors.lastname: Rothery
authors.forename: Janette M
authors.initials: JM
authors.lastname: Hudson
authors.forename: Anne K
authors.initials: AK
authors.lastname: Jones
authors.forename: Caroline S
authors.initials: CS
authors.lastname: Miles
authors.forename: Malcolm D
authors.initials: MD
authors.lastname: Walkinshaw
authors.forename: Fraser A
authors.initials: FA
authors.lastname: Armstrong
authors.forename: Graeme A
authors.initials: GA
authors.lastname: Reid
authors.forename: Stephen K
authors.initials: SK
authors.lastname: Chapman
mesh_terms: D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D049668:History, 15th Century; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020592:Shewanella / Q000201:enzymology*; D013385:Succinate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D013385:Succinate Dehydrogenase