TU Darmstadt / ULB / TUbiblio

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

Lancaster, C. R. D. and Sauer, U. S. and Gross, R. and Haas, A. H. and Graf, J. and Schwalbe, H. and Mäntele, W. and Simon, J. and Madej, M. G. (2005):
Experimental support for the "E pathway hypothesis" of coupled transmembrane e- and H+ transfer in dihemic quinol:fumarate reductase.
In: Proceedings of the National Academy of Sciences of the United States of America, pp. 18860-5, 102, (52), ISSN 0027-8424,
[Article]

Abstract

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.

Item Type: Article
Erschienen: 2005
Creators: Lancaster, C. R. D. and Sauer, U. S. and Gross, R. and Haas, A. H. and Graf, J. and Schwalbe, H. and Mäntele, W. and Simon, J. and Madej, M. G.
Title: Experimental support for the "E pathway hypothesis" of coupled transmembrane e- and H+ transfer in dihemic quinol:fumarate reductase.
Language: English
Abstract:

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.

Journal or Publication Title: Proceedings of the National Academy of Sciences of the United States of America
Volume: 102
Number: 52
Divisions: 10 Department of Biology > Microbial Energy Conversion and Biotechnology
?? fb10_mikrobiologie ??
10 Department of Biology
Date Deposited: 07 Dec 2010 15:18
Export:
Suche nach Titel in: TUfind oder in Google

Optionen (nur für Redakteure)

View Item View Item