Experimental support for the "E pathway hypothesis" of coupled transmembrane e- and H+ transfer in dihemic quinol:fumarate reductase.
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.
Lancaster
C Roy D
CR
Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max-von-Laue-Strasse 3, D-60438 Frankfurt am Main, Germany. roy.lancaster@mpibp-frankfurt.mpg.de
Sauer
Ursula S
US
Gross
Roland
R
Haas
Alexander H
AH
Graf
Jürgen
J
Schwalbe
Harald
H
Mäntele
Werner
W
Simon
Jörg
J
Madej
M Gregor
MG
D002462:Cell Membrane / Q000378:metabolism*; D018360:Crystallography, X-Ray; D004563:Electrochemistry; D004579:Electron Transport; D004583:Electrons; D018698:Glutamic Acid / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000737:chemistry; D007700:Kinetics; D008564:Membrane Potentials; D008954:Models, Biological; D008956:Models, Chemical; D008958:Models, Molecular; D015233:Models, Statistical; D008962:Models, Theoretical; D008968:Molecular Conformation; D009285:Naphthoquinones / Q000737:chemistry; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism; D010100:Oxygen / Q000737:chemistry; D011487:Protein Conformation; D011522:Protons; D012997:Solvents / Q000737:chemistry; D013053:Spectrophotometry; D016968:Wolinella / Q000378:metabolism
D009285:Naphthoquinones; D011522:Protons; D012997:Solvents; D018698:Glutamic Acid; C063002:2,3-dimethoxy-1,4-naphthoquinone; D010088:Oxidoreductases; C424907:quinol fumarate reductase; D010100:Oxygen