Publication
527
Biochem., 40 (5), 1248 -1256, 2001
DOI: 10.1021/bi002325y S0006-2960(00)02325-4 |
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Rate Constants in Two Dimensions of Electron Transfer
between Pyruvate Oxidase, a Membrane Enzyme, and Ubiquinone (Coenzyme
Q8),its water-insoluble electron carrier
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Damien Marchal, Jacques Pantigny, Jean Marc Laval, Jacques Moiroux, and Christian Bourdillon
Laboratoire de Technologie Enzymatique, Unité associée au CNRS No. 6022,
Université de Technologie de Compiègne, B.P. 20529, 60205 Compiègne Cedex,
France, Laboratoire d'Electrochimie Moléculaire, Unité associée au CNRS No.
7591, Université Paris, 7-Denis Diderot, 75251 Paris Cedex 05, France
The functionality of the membrane-bound, ubiquinone-dependent pyruvate
oxidase from the respiratory chain of Escherichia coli was
reconstituted with a supported lipidic structure. The artificial structure was
especially designed to allow the electrochemical control of the quinone pool
through the lateral mobility of the ubiquinone (Q8) molecules. The
kinetic coupling of the enzyme bound to the lipid structure with the quinone
pool was ensured by the regeneration of the oxidized form of ubiquinone at the
electrochemical interface. Such an experimental approach enabled us to carry out
an unprecedented determination of the kinetic parameters controlling the
reaction between the enzyme bound and the electron carrier under conditions
taking rigorously into account the fact that the freedom of motion is restricted
to two dimensions. The kinetic constants we found show that the activated enzyme
can be efficiently regulated by the oxidation level of the quinone pool in
natural membranes. |