Lancaster, C. R. D. ; Sauer, U. S. ; Gross, R. ; Haas, A. H. ; Graf, J. ; Schwalbe, H. ; Mäntele, W. ; Simon, J. ; 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, 102 (52)
Artikel, Bibliographie
Kurzbeschreibung (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.
| Typ des Eintrags: | Artikel |
|---|---|
| Erschienen: | 2005 |
| Autor(en): | Lancaster, C. R. D. ; Sauer, U. S. ; Gross, R. ; Haas, A. H. ; Graf, J. ; Schwalbe, H. ; Mäntele, W. ; Simon, J. ; Madej, M. G. |
| Art des Eintrags: | Bibliographie |
| Titel: | Experimental support for the "E pathway hypothesis" of coupled transmembrane e- and H+ transfer in dihemic quinol:fumarate reductase. |
| Sprache: | Englisch |
| Publikationsjahr: | 2005 |
| Titel der Zeitschrift, Zeitung oder Schriftenreihe: | Proceedings of the National Academy of Sciences of the United States of America |
| Jahrgang/Volume einer Zeitschrift: | 102 |
| (Heft-)Nummer: | 52 |
| Kurzbeschreibung (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. |
| Fachbereich(e)/-gebiet(e): | 10 Fachbereich Biologie > Microbial Energy Conversion and Biotechnology ?? fb10_mikrobiologie ?? 10 Fachbereich Biologie |
| Hinterlegungsdatum: | 07 Dez 2010 15:18 |
| Letzte Änderung: | 05 Mär 2013 09:42 |
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