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Journal/Book: Proc. Natl. Acad. Sci. USA
Published: 2005
Pages: 18860–18865
Volume: 102
Accession no.: 103

Experimental support for the “E pathway hypothesis” of coupled transmembrane e- and H+ transfer in dihemic quinol:fumarate reductase.

C. Roy D Lancaster, Ursula S Sauer, Roland Gross, Alexander H Haas, Jürgen Graf, Harald Schwalbe, Werner Mäntele, Jörg Simon, M. Gregor Madej
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.
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Prof. Dr. Harald Schwalbe
Institut für Organische Chemie und Chemische Biologie
Johann Wolfgang Goethe Universität
Max-von-Laue-Str. 7
D-60438 Frankfurt am Main
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