Microsecond Time-Resolved Absorption Spectroscopy Used to Study CO Compounds of Cytochrome bd from Escherichia coli

Cytochrome bd is a tri-heme (b558, b595, d) respiratory oxygen reductase that is found in many bacteria including pathogenic species. It couples the electron transfer from quinol to O2 with generation of an electrochemical proton gradient. We examined photolysis and subsequent recombination of CO with isolated cytochrome bd from Escherichia coli in oneelectron reduced (MV) and fully reduced (R) states by microsecond time-resolved absorption spectroscopy at 532-nm excitation. Both Soret and visible band regions were examined. CO photodissociation from MV enzyme possibly causes fast (t,1.5 ms) electron transfer from heme d to heme b595 in a small fraction of the protein, not reported earlier. Then the electron migrates to heme b558 (t,16 ms). It returns from the b-hemes to heme d with t,180 ms. Unlike cytochrome bd in the R state, in MV enzyme the apparent contribution of absorbance changes associated with CO dissociation from heme d is small, if any. Photodissociation of CO from heme d in MV enzyme is suggested to be accompanied by the binding of an internal ligand (L) at the opposite side of the heme. CO recombines with heme d (t,16 ms) yielding a transient hexacoordinate state (CO-Fe2+ -L). Then the ligand slowly (t,30 ms) dissociates from heme d. Recombination of CO with a reduced heme b in a fraction of the MV sample may also contribute to the 30-ms phase. In R enzyme, CO recombines to heme d (t,20 ms), some heme b558 (t,0.2–3 ms), and finally migrates from heme d to heme b595 (t,24 ms) in ,5% of the enzyme population. Data are consistent with the recent nanosecond study of Rappaport et al. conducted on the membranes at 640-nm excitation but limited to the Soret band. The additional phases were revealed due to differences in excitation and other experimental conditions

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 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-g 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 (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.</p

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 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-g 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 (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.</p