Characterization of cytochrome bo3 activity in a native-like surface-tethered membrane.
We have developed a simple native-like surface-tethered membrane system to investigate the activity of cbo(3) (cytochrome bo(3)), a terminal oxidase in Escherichia coli. The tethered membranes consist of E. coli inner-membrane extracts mixed with additional E. coli lipids containing various amounts of the cbo(3) substrate UQ-10 (ubiquinol-10). Tethered membranes are formed by self-assembly from vesicles on to gold electrodes functionalized with cholesterol derivatives. cbo(3) activity was monitored using CV (cyclic voltammetry) with electron transfer to cbo(3) mediated by UQ-10. The apparent K(m) for oxygen with this system is 1.1+/-0.4 microM, in good agreement with values reported in the literature for whole-cell experiments and for purified cbo(3). Increasing the concentration of lipophilic UQ-10 in the membrane leads to an increase in cbo(3) activity. The activity of cbo(3) with long-chain ubiquinones appears to be different from previous reports using short-chain substrate analogues such as UQ-1 in that typical Michaelis-Menten kinetics are not observed using UQ-10. This native-like membrane model thus provides new insights into the interaction of transmembrane enzymes with hydrophobic substrates which contrasts with studies using hydrophilic UQ analogues.
Weiss
Sophie A
SA
School of Physics and Astronomy, University of Leeds, Leeds, U.K.
Bushby
Richard J
RJ
Evans
Stephen D
SD
Henderson
Peter J F
PJ
Jeuken
Lars J C
LJ
D002462:Cell Membrane / Q000201:enzymology*; D003573:Cytochrome b Group; D003580:Cytochromes / Q000235:genetics / Q000378:metabolism*; D004563:Electrochemistry; D004566:Electrodes; D004789:Enzyme Activation; D029968:Escherichia coli Proteins; D007700:Kinetics; D010100:Oxygen / Q000378:metabolism
D003573:Cytochrome b Group; D003580:Cytochromes; D029968:Escherichia coli Proteins; C083850:cytochrome bo3, E coli; D010100:Oxygen