2bs3: Difference between revisions

Revision as of 17:41, 21 February 2008

GLU C180-> GLN VARIANT QUINOL:FUMARATE REDUCTASE FROM WOLINELLA SUCCINOGENES

OverviewOverview

Reconciliation of apparently contradictory experimental results obtained on the quinol:fumarate reductase, a diheme-containing respiratory membrane protein complex from Wolinella succinogenes, was previously obtained by the proposal of the so-called "E pathway hypothesis." According to this hypothesis, transmembrane electron transfer via the heme groups is strictly coupled to cotransfer of protons via a transiently established pathway thought to contain the side chain of residue Glu-C180 as the most prominent component. Here we demonstrate that, after replacement of Glu-C180 with Gln or Ile by site-directed mutagenesis, the resulting mutants are unable to grow on fumarate, and the membrane-bound variant enzymes lack quinol oxidation activity. Upon solubilization, however, the purified enzymes display approximately 1/10 of the specific quinol oxidation activity of the wild-type enzyme and unchanged quinol Michaelis constants, K(m). The refined x-ray crystal structures at 2.19 A and 2.76 A resolution, respectively, rule out major structural changes to account for these experimental observations. Changes in the oxidation-reduction heme midpoint potential allow the conclusion that deprotonation of Glu-C180 in the wild-type enzyme facilitates the reoxidation of the reduced high-potential heme. Comparison of solvent isotope effects indicates that a rate-limiting proton transfer step in the wild-type enzyme is lost in the Glu-C180 --> Gln variant. The results provide experimental evidence for the validity of the E pathway hypothesis and for a crucial functional role of Glu-C180.

About this StructureAbout this Structure

2BS3 is a Protein complex structure of sequences from Wolinella succinogenes with , , , , , , and as ligands. Active as Succinate dehydrogenase, with EC number 1.3.99.1 Known structural/functional Site: . Full crystallographic information is available from OCA.

ReferenceReference

Experimental support for the "E pathway hypothesis" of coupled transmembrane e- and H+ transfer in dihemic quinol:fumarate reductase., Lancaster CR, Sauer US, Gross R, Haas AH, Graf J, Schwalbe H, Mantele W, Simon J, Madej MG, Proc Natl Acad Sci U S A. 2005 Dec 27;102(52):18860-5. PMID:16380425

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 ==Overview==
-Reconciliation of apparently contradictory experimental results obtained, on the quinol:fumarate reductase, a diheme-containing respiratory membrane, protein complex from Wolinella succinogenes, was previously obtained by, the proposal of the so-called "E pathway hypothesis." According to this, hypothesis, transmembrane electron transfer via the heme groups is, strictly coupled to cotransfer of protons via a transiently established, pathway thought to contain the side chain of residue Glu-C180 as the most, prominent component. Here we demonstrate that, after replacement of, Glu-C180 with Gln or Ile by site-directed mutagenesis, the resulting, mutants are unable to grow on fumarate, and the membrane-bound variant, enzymes lack quinol oxidation activity. Upon solubilization, however, the, purified enzymes display approximately 1/10 of the specific quinol, oxidation activity of the wild-type enzyme and unchanged quinol Michaelis, constants, K(m). The refined x-ray crystal structures at 2.19 A and 2.76 A, resolution, respectively, rule out major structural changes to account for, these experimental observations. Changes in the oxidation-reduction heme, midpoint potential allow the conclusion that deprotonation of Glu-C180 in, the wild-type enzyme facilitates the reoxidation of the reduced, high-potential heme. Comparison of solvent isotope effects indicates that, a rate-limiting proton transfer step in the wild-type enzyme is lost in, the Glu-C180 --&gt; Gln variant. The results provide experimental evidence, for the validity of the E pathway hypothesis and for a crucial functional, role of Glu-C180.
+Reconciliation of apparently contradictory experimental results obtained on the quinol:fumarate reductase, a diheme-containing respiratory membrane protein complex from Wolinella succinogenes, was previously obtained by the proposal of the so-called "E pathway hypothesis." According to this hypothesis, transmembrane electron transfer via the heme groups is strictly coupled to cotransfer of protons via a transiently established pathway thought to contain the side chain of residue Glu-C180 as the most prominent component. Here we demonstrate that, after replacement of Glu-C180 with Gln or Ile by site-directed mutagenesis, the resulting mutants are unable to grow on fumarate, and the membrane-bound variant enzymes lack quinol oxidation activity. Upon solubilization, however, the purified enzymes display approximately 1/10 of the specific quinol oxidation activity of the wild-type enzyme and unchanged quinol Michaelis constants, K(m). The refined x-ray crystal structures at 2.19 A and 2.76 A resolution, respectively, rule out major structural changes to account for these experimental observations. Changes in the oxidation-reduction heme midpoint potential allow the conclusion that deprotonation of Glu-C180 in the wild-type enzyme facilitates the reoxidation of the reduced high-potential heme. Comparison of solvent isotope effects indicates that a rate-limiting proton transfer step in the wild-type enzyme is lost in the Glu-C180 --&gt; Gln variant. The results provide experimental evidence for the validity of the E pathway hypothesis and for a crucial functional role of Glu-C180.
 ==About this Structure==
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 [[Category: Succinate dehydrogenase]]
 [[Category: Wolinella succinogenes]]
-[[Category: Lancaster, C.R.D.]]
+[[Category: Lancaster, C R.D.]]
 [[Category: CIT]]
 [[Category: F3S]]
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 [[Category: tricarboxylic acid cycle]]
-''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Sun Feb  3 10:27:18 2008''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 16:41:03 2008''