Refine
Document Type
- Article (1)
- Doctoral Thesis (1)
Language
- English (2)
Has Fulltext
- yes (2)
Is part of the Bibliography
- no (2)
Institute
Succinate:quinone oxidoreductases (SQORs) are integral membrane protein complexes, which couple the two-electron oxidation of succinate to fumarate (succinate → fumarate + 2H+ + 2e-) to the two-electron reduction of quinone to quinol (quinone + 2H+ + 2e- → quinol) as well as catalyzing the opposite reaction, the reduction of fumarate by quinol. In mitochondria and some aerobic bacteria, succinate:ubiquinone reductase, also known as complex II of the aerobic respiratory chain or as succinate dehydrogenase from the tricarboxylic acid (TCA or Krebs) cycle, catalyzes the oxidation of succinate by ubiquinone, which is mildly exergonic under standart conditions and not directly associated with energy storage in the form of a transmembrane electrochemical proton potential (Δp). Gram-positive bacteria do not contain ubiquinone but rather menaquinone, a quinone with significantly lower oxidation-reduction (“redox”) midpoint potential. In these cases, the catalyzed oxidation of succinate by quinone is endergonic under standard conditions. Consequently, these bacteria face a thermodynamic problem in supporting the catalysis of this reaction in vivo. Based on experimental evidence obtained on whole cells and purified membranes, it had previously been proposed that the SQR from Gram-positive bacteria supports this reaction at the expense of the protonmotive force, Δp. Nonetheless, it has been argued that the observed Δp dependence is not associated specifically with the activity of SQR because the occurrence of artifacts in experiments with bacterial membranes and whole cells can not be fully excluded. Clearly, definitive insight into the mechanism of catalysis of this intriguing reaction required a corresponding functional characterization of an isolated, membranebound SQR from a Gram-positive bacterium. The first aim of the present work addresses the question if the general feasibility of the energetically uphill electron transfer from succinate to menaquinone is associated specifically to a single enzyme complex, the SQR. The prerequisite to achieve this goal was stable preparation of this enzyme.
Bacterial sugar symporters in the Major Facilitator Superfamily (MFS) use the H+ (and in a few cases Na+) electrochemical gradients to achieve active transport of sugar into the cell. Because a number of structures of MFS sugar symporters have been solved recently, molecular insight into the transport mechanism is possible from detailed functional analysis. We present here a comparative electrophysiological study of the lactose permease (LacY), the fucose permease (FucP) and the xylose permease (XylE), which reveals common mechanistic principles and differences. In all three symporters energetically downhill electrogenic sugar/H+ symport is observed. Comparison of the pH dependence of symport at symmetrical pH exhibits broad bell-shaped pH profiles extending over 3 to 6 pH units and a decrease at extremely alkaline pH ≥ 9.4 and at acidic to neutral pH = 4.6–7.5. The pH dependence can be described by an acidic to neutral apparent pK (pKapp) and an alkaline pKapp. Experimental evidence suggests that the alkaline pKapp is due to H+ depletion at the protonation site, while the acidic pKapp is due to inhibition of deprotonation. Since previous studies suggest that a single carboxyl group in LacY (Glu325) may be the only side chain directly involved in H+ translocation and a carboxyl side chain with similar properties has been identified in FucP (Asp46) and XylE (Asp27), the present results imply that the pK of this residue is switched during H+/sugar symport in all three symporters.