Identification of a stable semiquinone intermediate in the purified and membrane bound ubiquinol oxidase-cytochrome bd from Escherichia coli.
The quinol oxidase, cytochrome bd, functions as a terminal oxidase in the Escherichia coli respiratory chain, reducing O2 to water and using ubiquinol-8 or menaquinol-8 as its immediate reductant. The oxidation of quinol is by the low-spin ferri-haem, cytochrome b558. This occurs at a quinol-binding site by sequential one electron steps, requiring the stabilisation of the semiquinone intermediate. We have observed, by EPR spectroscopy, the properties of this semiquinone radical in appropriately poised samples of purified enzyme reconstituted with excess of ubiquinone-8 and menaquinone-8 analogues. The line width of the EPR spectrum is approximately 0.9 mT, which is consistent with a semiquinone anion of this type. The line shape is Gaussian. The semiquinone is highly stabilised with respect to free semiquinone; significant free radical can be observed at pH 7.0 and above. The pH dependence of the redox reactions indicate that the anionic form of the semiquinone and the neutral form of the quinol predominate in the pH range studied. The pH dependence of the mid-point potentials of the one electron reactions from pH 7.0-9.0 is 120 mV/pH change for the semiquinone anion to quinol (E2) and none for the quinone to semiquinone (E1). The semiquinone radical is attenuated on titration with putative inhibitors of this quinone-binding site. We have similarly studied the semiquinone in membrane preparations from a strain with overexpression of cytochrome bd oxidase. The data can be fitted with the assumption of a single quinone-binding site.
School of Biological and Medical Sciences, University of St Andrews, Scotland, UK.
S F
SF
Hastings
T M
TM
Kaysser
F
F
Jiang
J C
JC
Salerno
R B
RB
Gennis
W J
WJ
Ingledew
D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D003573:Cytochrome b Group; D003580:Cytochromes / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport; D045222:Electron Transport Chain Complex Proteins; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000378:metabolism; D008956:Models, Chemical; D009573:Nitrites / Q000378:metabolism; D010088:Oxidoreductases / Q000378:metabolism*; D011199:Potentiometry; D012995:Solubility; D013816:Thermodynamics; D014451:Ubiquinone / Q000031:analogs & derivatives*; D014812:Vitamin K / Q000031:analogs & derivatives*
D001426:Bacterial Proteins; D003573:Cytochrome b Group; D003580:Cytochromes; D045222:Electron Transport Chain Complex Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D009573:Nitrites; D014812:Vitamin K; D014451:Ubiquinone; D010088:Oxidoreductases; C040077:cytochrome bd terminal oxidase complex, E coli