A proton delivery pathway in the soluble fumarate reductase from Shewanella frigidimarina.
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Pankhurst KL, Mowat CG, Rothery EL, Hudson JM, Jones AK, Miles CS, Walkinshaw MD, Armstrong FA, Reid GA, Chapman SK
A proton delivery pathway in the soluble fumarate reductase from Shewanella frigidimarina.
J Biol Chem. 2006 Jul 21;281(29):20589-97. Epub 2006 May 12.
- PubMed ID
- 16699170 [ View in PubMed]
- 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.