2bs3: Difference between revisions
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==Overview== | ==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, ... | 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 Structure== | ==About this Structure== | ||
2BS3 is a | 2BS3 is a [http://en.wikipedia.org/wiki/Protein_complex Protein complex] structure of sequences from [http://en.wikipedia.org/wiki/Wolinella_succinogenes Wolinella succinogenes] with NA, FAD, CIT, FES, F3S, SF4, HEM and LMT as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Succinate_dehydrogenase Succinate dehydrogenase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.3.99.1 1.3.99.1] Structure known Active Site: AC1. Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=2BS3 OCA]. | ||
==Reference== | ==Reference== | ||
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[[Category: tricarboxylic acid cycle]] | [[Category: tricarboxylic acid cycle]] | ||
''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on | ''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Mon Nov 5 14:09:13 2007'' | ||