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The measurement of respiratory chain enzyme activities is an integral part of basic research as well as for specialized examinations in clinical biochemistry. Most of the enzymes use ubiquinone as one of their substrates. For current in vitro measurements, several hydrophilic analogues of native ubiquinone are used depending on the enzyme and the w...
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... verify the general validity of our results, we applied Coenzyme Q 2 to measure activities of NADH dehydrogenase, succinate dehydrogenase and cytochrome c reductase in both mitochondrial lysates of S. cerevisiae and chicken liver ( Table 2). The values of measured activities in both organisms were roughly comparable with those obtained for the trypanosomatids investigated in this study. ...Citations
... In connection with the question above, Cermáková et al. (35) reported that T. brucei AOX strictly recognizes the isoprene unit in the side chain of UQs; for example, the electron transfer activity of a reduced form of DB is much poorer than that of reduced UQ2 and UQ1 (activity order of UQ2H2 > UQ1H2 >> DBH2). We confirmed this tendency for the three quinols using our AOX preparation ( Figure 7). ...
NADH-quinone oxidoreductase (complex I) couples electron transfer from NADH to quinone with proton translocation across the membrane. Quinone reduction is a key step for energy transmission from the site of quinone reduction to the remotely located proton-pumping machinery of the enzyme. Although structural biology studies have proposed the existence of a long and narrow quinone-access channel, the physiological relevance of this channel remains debatable. We investigated here whether complex I in bovine heart submitochondrial particles (SMPs) can catalytically reduce a series of oversized ubiquinones (OS-UQs), which are highly unlikely to transit the narrow channel because their side chain includes a bulky “block” that is ~13 Å across. We found that some OS-UQs function as efficient electron acceptors from complex I, accepting electrons with an efficiency comparable to ubiquinone-2. The catalytic reduction and proton translocation coupled with this reduction were completely inhibited by different quinone-site inhibitors, indicating that the reduction of OS-UQs takes place at the physiological reaction site for ubiquinone. Notably, the proton-translocating efficiencies of OS-UQs significantly varied depending on their side-chain structures, suggesting that the reaction characteristics of OS-UQs affect the predicted structural changes of the quinone reaction site required for triggering proton translocation. These results are difficult to reconcile with the current channel model; rather, the access path for ubiquinone may be open to allow OS-UQs to access the reaction site. Nevertheless, contrary to the observations in SMPs, OS-UQs were not catalytically reduced by isolated complex I reconstituted into liposomes. We discuss possible reasons for these contradictory results.
The Na⁺-pumping NADH-ubiquinone (UQ) oxidoreductase (Na⁺-NQR) is an essential bacterial respiratory enzyme that generates a Na⁺ gradient across the cell membrane. However, the mechanism that couples the redox reactions to Na⁺ translocation remains unknown. To address this, we examined the relation between reduction of UQ and Na⁺ translocation using a series of synthetic UQs with Vibrio cholerae Na⁺-NQR reconstituted into liposomes. UQ0 that has no side chain and UQCH3 and UQC2H5, which have methyl and ethyl side chains, respectively, were catalytically reduced by Na⁺-NQR, but their reduction generated no membrane potential, indicating that the overall electron transfer and Na⁺ translocation are not coupled. While these UQs were partly reduced by electron leak from the cofactor(s) located upstream of riboflavin, this complete loss of Na⁺ translocation cannot be explained by the electron leak. Lengthening the UQ side chain to n-propyl (C3H7) or longer significantly restored Na⁺ translocation. It has been considered that Na⁺ translocation is completed when riboflavin, a terminal redox cofactor residing within the membrane, is reduced. In this view, the role of UQ is simply to accept electrons from the reduced riboflavin to regenerate the stable neutral riboflavin radical and reset the catalytic cycle. However, the present study revealed that the final UQ reduction via reduced riboflavin makes an important contribution to Na⁺ translocation through a critical role of its side chain. Based on the results, we discuss the critical role of the UQ side chain in Na⁺ translocation.
Complex I (NADH dehydrogenase) is the first enzyme in the respiratory chain. It catalyses the electron transfer from NADH to ubiquinone that is associated with proton pumping out of the matrix. In this study, we characterized NADH dehydrogenase activity in seven monoxenous trypanosomatid species: Blechomonas ayalai , Herpetomonas tarakana , Kentomonas sorsogonicus , Leptomonas seymouri , Novymonas esmeraldas , Sergeia podlipaevi and Wallacemonas raviniae . We also investigated the subunit composition of the complex I in dixenous Phytomonas serpens , in which its presence and activity have been previously documented. In addition to P. serpens , the complex I is functionally active in N. esmeraldas and S. podlipaevi . We also identified 24–32 subunits of the complex I in individual species by using mass spectrometry. Among them, for the first time, we recognized several proteins of the mitochondrial DNA origin.
