Identification of a stable semiquinone intermediate in the purified and membrane bound ubiquinol oxidase‐cytochrome bd from Escherichia coli

Abstract

The quinol oxidase, cytochrome bd, functions as a terminal oxidase in the Escherichia coli respiratory chain, reducing O 2 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 b 558. 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 7.0Ϫ9.0 is 120 mV/pH change for the semiquinone anion to quinol (E 2) and none for the quinone to semiquinone (E 1 ). 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.Keywords : cytochrome bd; quinol oxidation; quinone-binding site; semiquinone stabilisation.Quinone-binding sites are obligatory in quinone-utilising re-mitochondria and chloroplasts. The respiratory chains of Escherichia coli contain them because the respiratory chain is termispiratory and photosynthetic complexes. In addition to their importance in catalysis in electron-transfer systems involved in en-nated by quinol oxidases ; cytochromes bo and bd, fumarate reductase, dimethylsulphoxide reductase and nitrate reductase. In ergy transduction, these loci are the site of action of numerous inhibitors including pesticides, herbicides and antibiotics [1].the Escherichia coli respiratory chain two quinones are employed, ubiquinone-8 and menaquinone-8. Most dehydrogenases Quinone-binding sites are of three types; reduction (acceptor), oxidation (donor) and pair-splitting loci. The site under con-and oxidases do not appear to discriminate markedly between them, although the relative concentrations of the two species are sideration here is an oxidation site as it supplies reducing equivalents to the low-spin b 558 of cytochrome bd [2] (but see also regulated and influenced by growth conditions [6].Mitochondria have acceptor loci associated with the NADH [3]); the potentials of the two half reactions thus have to be approximately equated as the electrons are fed to the same and succinate dehydrogenases. They also have two sites associated with complex III and involved in the proton-motive...

Anaerobic Respiration of Escherichia coli in the Mouse Intestine

“…However, the midpoint potential of UQ is best suited for aerobic respiration, whereas MQ and DMQ are involved primarily in anaerobic respiration, with DMQ having a midpoint potential between those of UQ and MQ (70). In general, anaerobic terminal reductases have higher activity with MQ and DMQ, cytochrome bo 3 has the highest activity with UQ, and cytochrome bd apparently uses both UQ and MQ (23). Electron flow to fumarate, TMAO, and nitrite is via both MQ and DMQ, while electron flow to DMSO is primarily via MQ.…”

Differences in Protonation of Ubiquinone and Menaquinone in Fumarate Reductase from Escherichia coli

“…EPR Studies of Semiquinone Radicals Bound to the FrdC E29L Mutant-It has been shown that E. coli bd-and bo-quinol oxidases, enzymes that interact with both UQ and MQ, are able to stabilize both radicals at the Q-binding site (26,38). Wild-type QFR does not show stabilized MQ radicals (17), as well as, UQ radical (data not shown).…”

“…Potentiometric titration data were analyzed by plotting the intensity of the g ϭ 2.00 peak-trough versus E h and fitting the resultant bell-shaped titration curve to two midpoint redox potential (E m ) values (26). E m values were representatives of two to three independent titrations with a standard deviation of ϳϮ10 mV.…”

Mass Spectrometric Analysis of the Ubiquinol-binding Site in Cytochrome bd from Escherichia coli

“…Because of auto-oxidation of low potential quinols, the K m values are not reported for menaquinol oxidases. Kinetic studies, electron paramagnetic resonance studies on semiquinone anion (53), and inhibitor binding studies (54) are consistent with the presence of a single quinol/quinone-binding site in cytochrome bd.…”