TY - JOUR
T1 - A proton delivery pathway in the soluble fumarate reductase from Shewanella frigidimarina
AU - Pankhurst, Katherine L.
AU - Mowat, Christopher G.
AU - Rothery, Emma L.
AU - Hudson, Janette M.
AU - Jones, Anne K.
AU - Miles, Caroline S.
AU - Walkinshaw, Malcolm D.
AU - Armstrong, Fraser A.
AU - Reid, Graeme A.
AU - Chapman, Stephen K.
PY - 2006/7/21
Y1 - 2006/7/21
N2 - 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 kcat values compared with the wildtype 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 Å 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.
AB - 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 kcat values compared with the wildtype 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 Å 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.
UR - http://www.scopus.com/inward/record.url?scp=33745972079&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=33745972079&partnerID=8YFLogxK
U2 - 10.1074/jbc.M603077200
DO - 10.1074/jbc.M603077200
M3 - Article
C2 - 16699170
AN - SCOPUS:33745972079
SN - 0021-9258
VL - 281
SP - 20589
EP - 20597
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 29
ER -