Article

The Kinetics of the Redox Reactions of Ubiquinone Related to the Electron-Transport Activity in the Respiratory Chain

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... Evidence in favor of the RCM derived from 3 major kinds of observations: (a) The integral proteins of the inner membrane are randomly distributed in the bilayer, and phospholipid dilution of the mitochondrial membrane proteins slows down electron transfer [Schneider et al., 1982]. (b) Electron transfer in the CoQ and cytochrome c region obeys pool behavior according to the equation developed for CoQ by Kröger and Klingenberg [1973]. (c) Electron transfer follows saturation kinetics with respect to CoQ and cytochrome c concentrations [Mac-Lennan et al., 1966;Estornell et al., 1992]. ...
... The notion of the CoQ pool as the mechanism for integrated electron transfer from dehydrogenases to cytochromes, described by the hyperbolic relationship between the observed rate of electron transfer of the entire respiratory chain and the rate of either reduction or oxidation of CoQ [Kröger and Klingenberg, 1973], has been widely accepted and is covered in all biochemistry textbooks. ...
... The CoQ pool is required for electron transfer from Complex II to Complex III; indeed Complex II kineti-cally follows pool behavior in mitochondria after extraction and reconstitution [Kröger and Klingenberg, 1973] with CoQ [Stoner, 1984] in accordance with the lack of supercomplexes found by both BN-PAGE and flux control analysis (see previous section). Furthermore, other enzymes such as glycerol-3-phosphate dehydrogenase, ETF dehydrogenase, dihydroorotate dehydrogenase, choline dehydrogenase, SQR, and proline dehydrogenase, which are present in lower amounts and are likely to be rate-limiting in an integrated electron transfer, are probably inserted in the respiratory chain by interaction through the CoQ pool [Genova and Lenaz, 2011]. ...
Article
Two alternative models of organization of the mitochondrial electron transport chain (mETC) have been alternatively favored or questioned by the accumulation evidences of different sources, the solid model or the random collision model. Both agree in the number of respiratory complexes (I-IV) that participate in the mETC, but while the random collision model proposes that Complexes I-IV do not interact physically and that electrons are transferred between them by coenzyme Q and cytochrome c, the solid model proposes that all complexes super-assemble in the so-called respirasome. Recently, the plasticity model has been developed to incorporate the solid and the random collision model as extreme situations of a dynamic organization, allowing super-assembly free movement of the respiratory complexes. In this review, we evaluate the supporting evidences of each model and the implications of the super-assembly in the physiological role of coenzyme Q.
... Coenzyme Q plays an important role as a mobile electron carrier in the respiratory chain function, and enhances cellular energy. 21 Idebenone is a synthetic analog of coenzyme Q 22 and has been reported to mitigate the reduction in ATP levels associated with mitochondrial dysfunction. [21][22][23][24][25] Mitochondrial β-oxidation is involved in energy production, and l-carnitine contributes to β-oxidation of quarried fatty acids from the mitochondrial membrane. ...
... 21 Idebenone is a synthetic analog of coenzyme Q 22 and has been reported to mitigate the reduction in ATP levels associated with mitochondrial dysfunction. [21][22][23][24][25] Mitochondrial β-oxidation is involved in energy production, and l-carnitine contributes to β-oxidation of quarried fatty acids from the mitochondrial membrane. 26 We therefore also assessed whether idebenone and l-carnitine could regulate the effects of 2-DG on the action of isoflurane anesthesia. ...
... Idebenone is an analog of coenzyme Q10 and has been reported to rescue ATP levels in mitochondrial dysfunction. [21][22][23][24][25] Next, we assessed whether idebenone was able to inhibit the effects of 2-DG on the anesthetic effects. The relationship curve between the percentage of mice with LORR and the concentrations of isoflurane was determined in the mice pretreated with idebenone plus saline or idebenone plus 2-DG. ...
Article
Full-text available
Background: The mechanisms of general anesthesia by volatile drugs remain largely unknown. Mitochondrial dysfunction and reduction in energy levels have been suggested to be associated with general anesthesia status. 2-Deoxy-D-glucose (2-DG), an analog of glucose, inhibits hexokinase and reduces cellular levels of adenosine triphosphate (ATP). 3-Nitropropionic acid is another compound which can deplete ATP levels. In contrast, idebenone and L-carnitine could rescue deficits of energy. We therefore sought to determine whether 2-DG and/or 3-nitropropionic acid can enhance the anesthetic effects of isoflurane, and whether idebenone and L-carnitine can reverse the actions of 2-DG. Methods: C57BL/6J mice (8 months old) received different concentrations of isoflurane with and without the treatments of 2-DG, 3-nitropropionic acid, idebenone, and L-carnitine. Isoflurane-induced loss of righting reflex (LORR) was determined in the mice. ATP levels in H4 human neuroglioma cells were assessed after these treatments. Finally, 31P-magnetic resonance spectroscopy was used to determine the effects of isoflurane on brain ATP levels in the mice. Results: 2-DG enhanced isoflurane-induced LORR (P = 0.002, N = 15). 3-Nitropropionic acid also enhanced the anesthetic effects of isoflurane (P = 0.005, N = 15). Idebenone (idebenone + saline versus idebenone + 2-DG: P = 0.165, N = 15), but not L-carnitine (L-carnitine + saline versus L-carnitine + 2-DG: P < 0.0001, N = 15), inhibited the effects of 2-DG on enhancing isoflurane-induced LORR in the mice, as evidenced by 2-DG not enhancing isoflurane-induced LORR in the mice pretreated with idebenone. Idebenone (idebenone + saline versus idebenone + 2-DG: P = 0.177, N = 6), but not L-carnitine (L-carnitine + saline versus L-carnitine + 2-DG: P = 0.029, N = 6), also mitigated the effects of 2-DG on reducing ATP levels in cells, as evidenced by 2-DG not decreasing ATP levels in the cells pretreated with idebenone. Finally, isoflurane decreased ATP levels in both cultured cells and mouse brains (β-ATP: P = 0.003, N = 10; β-ATP/phosphocreatine: P = 0.006, N = 10; β-ATP/inorganic phosphate: P = 0.001, N = 10). Conclusions: These results from our pilot studies have established a system and generated a hypothesis that 2-DG enhances anesthetic effects via reducing energy levels. These findings should promote further studies to investigate anesthesia mechanisms.
... The overall respiratory chain activity was postulated as a sequen- tial transfer of electrons between four major multi-enzymatic complexes dispersed in the inner mitochondrial membrane (IMM): NADH dehydrogenase:ubiquinone oxidoreductase (com- plex I, CI), succinate:ubiquinone oxidoreductase (complex II, CII), ubiquinol:cytochrome c oxidoreductase or cytochrome bc1 com- plex (complex III, CIII), and cytochrome c oxidase (complex IV, CIV). In addition, the electron transfer was ensured by the diffusion of two mobile components acting as co-substrates: the lipophilic ubiquinone, also designated as coenzyme Q (CoQ), embedded in the membrane lipid bilayer, and the hydrophilic heme protein cytochrome c (cytc) located on the external surface of the IMM [3,4]. Altogether, these components form the mitochondrial respiratory chain (MRC) where cellular respiration takes place. ...
... The general vision gradually evolved into a "random colli- sion", "fluid" or "liquid-state" model, proposed by Hackenbrock and co-workers, that pictured all membrane proteins and redox compo- nents that catalyse electron transport and ATP synthesis in constant and independent diffusional motion, where electron transfer to place through diffusion-based collisions among the redox part- ners [11]. Evidence in favour of the "liquid-state model" arose from kinetic analyses proposing not only that electron transfer in the CoQ and cytc regions obeyed a pool behaviour in mammalian mitochon- dria, but also that it followed saturation kinetics with regards to CoQ and cytc concentrations [3,12]. Furthermore, enzymatic activities were retained upon isolation of the individual OXPHOS complexes [2] and the use of fluorescent antibodies against CIII 2 and CIV caused independent aggregation of these complexes, suggesting that they diffuse laterally in the membrane plane independent of one another [13]. ...
... In studies on Q-pool behavior in the respiratory chain [39] recently confirmed by others [40], linear dependence of NADH oxidase activity of bovine heart SMP on the content (concentration) of oxidized ubiquinone have been demonstrated. If extrapolated to the state where ubiquinone is fully oxidized, the data would correspond to turnover number of complex I 2 The mitochondrial enzyme exhibits complex kinetics in the quinone reductase activity due to slow transition between catalytically inert D-and active A-forms (operationally called the A/D-transition). ...
... 7 of about 500 sec -1 (pH 7.4, 25°C), a value that is substantially higher than those (averaged) measured at saturating concentrations of water-soluble quinones or in steady-state NADH oxidase assays (~250 sec -1 , see above). This is not surprising because during the steady state, fully uncoupled NADH oxidase operates at Q red /Q ox ratio of about 1. Linear, not hyperbolic, dependence of the NADH oxidase rate on molar fraction of oxidized ubiquinone (Q ox / (Q ox +Q red )) has been demonstrated [39]. Such dependence is expected either if the total concentration of quinone is much lower than the apparent K m for Q ox or if Q red competes with Q ox for binding at the reactive site with similar affinity. ...
Article
Kinetic characteristics of the proton-pumping NADH:quinone reductases (respiratory complexes I) are reviewed. Unsolved problems of the redox-linked proton translocation activities are outlined. The parameters of complex I-mediated superoxide/hydrogen peroxide generation are summarized, and the physiological significance of mitochondrial ROS production is discussed. This article is part of a Special Issue entitled Respiratory complex I, edited by Volkerzickermann and Ulrich Brandt.
... The role of isoprenoid quinones in the bioenergetics of cells was first proposed in the late 1950s (58). The observation that extraction of quinones from bioenergetic membranes inhibited the activity of mitochondrial complexes led to the conclusion that quinones are responsible for shuttling electrons between respiratory complexes (92,146,196). Further experiments revealed that semiquinone intermediate is formed during the catalytic cycle of cytochrome bc 1 (265), and this observation was adopted by Peter Michell to propose the mechanism of protonmotive Q cycle (177,178). ...
... Early experiments on the kinetics of electron transfer revealed that the Q pool creates rather homogeneous ensemble of molecules rapidly diffusing within lipid bilayer, being in equilibrium with binding sites of proteins (105,146). An alternative view assumes the compartmentalization of quinone molecules into separate pools within the membrane (107). ...
Article
Full-text available
Mitochondrial respiration, an important bioenergetic process, relies on operation of four membranous enzymatic complexes linked functionally by mobile, freely diffusible elements: quinone molecules in the membrane and water-soluble cytochromes c in the intermembrane space. One of the mitochondrial complexes, complex III (cytochrome bc1 or ubiquinol:cytochrome c oxidoreductase), provides an electronic connection between these two diffusible redox pools linking in a fully reversible manner two-electron quinone oxidation/reduction with one-electron cytochrome c reduction/oxidation. Several features of this homodimeric enzyme implicate that in addition to its well-defined function of contributing to generation of proton-motive force, cytochrome bc1 may be a physiologically important point of regulation of electron flow acting as a sensor of the redox state of mitochondria that actively responds to changes in bioenergetic conditions. These features include the following: the opposing redox reactions at quinone catalytic sites located on the opposite sides of the membrane, the inter-monomer electronic connection that functionally links four quinone binding sites of a dimer into an H-shaped electron transfer system, as well as the potential to generate superoxide and release it to the intermembrane space where it can be engaged in redox signaling pathways. Here we highlight recent advances in understanding how cytochrome bc1 may accomplish this regulatory physiological function, what is known and remains unknown about catalytic and side reactions within the quinone binding sites and electron transfers through the cofactor chains connecting those sites with the substrate redox pools. We also discuss the developed molecular mechanisms in the context of physiology of mitochondria. Copyright © 2015 the American Physiological Society.
... The essential role of CoQ in the function of the electron transport chain was confirmed with experiments involving reincorporation of the quinone into CoQ-depleted submitochondrial particles [Norling et al., 1974]. Oxidation-reduction cycling of CoQ during electron transport has been observed directly [Kroger and Klingenberg, 1973]. CoQ thus allows reversible interactions between the NADH dehydrogenase, succinate dehydrogenase, and cytochrome bci portions of the electron transport chain [Ernster, 1962;Chance and Hollunger, 1961;Low and Vallin, 1963]. ...
... There are currently two schools of thought about the distribution of CoQ in the mitochondrial membrane. One school subscribes to the view that CoQ exists as a freely diffusable homogenous pool, shuttling electrons between different complexes [Kroger and Klingenberg, 1973;Lenaz et al., 1997;Gupte et al., 1984]. The other view, based on the variations in the reducibility of CoQ by different substrates [Lass and Sohal, 1998;Jorgensen et al., 1985], is that CoQ is distributed heterogeneously, forming separate and distinct pools within the hydrophobic domain of the membranes. ...
Thesis
Mitochondrial complex I is a protein of 800 kDa consisting of 41 subunits. It contains flavin mononucleotide (FMN) and between 22-24 iron-sulphur (Fe-S) centres. Complex I is responsible for the oxidation of NADH to NAD+ in the presence of ubiquinone (CoQ) as electron acceptor. In this process, CoQ is reduced to ubiquinol (C0 QH2) which is re-oxidised to CoQ by complex III. Recently, it was shown in intact cells that prolonged exposure to nitric oxide (NO) results in a persistent inhibition of complex I activity, which is preceded by a decrease in the concentration of intracellular glutathione. The objective of this study was to investigate the possible mechanism(s) of the inhibition of complex I by NO using purified complex I from bovine heart mitochondria. The oxidation rate of NADH in the presence of complex I and CoQ was measured as an indicator of complex I activity, and the effect of different NO donors and incubation times on complex I activity was evaluated. Incubation of purified complex I with NO donors revealed no differences in the oxidation rate of NADH as compared to untreated controls. These results suggest that NO does not affect the activity of purified complex I. However, it was found that NADH added serially could be repeatedly oxidised by purified complex I and CoQ. In these conditions, exposure of CoQ to NO resulted in a reduction of the initial NADH oxidation and prevented almost completely the subsequent oxidation. Interestingly, the inhibitory effect of NO on CoQ was temporary and was completely reversible with time, suggesting the formation of a labile NO-CoQ adduct. Further biochemical and pharmacological analysed provided evidence of a chemical reaction between NO and CoQ.
... The functional significance of a random distribution of mitochondrial respiratory complexes had been supported in the past by the kinetic analysis of Kröger and Klingenberg for the enzymes connected by CoQ [4]. Kröger and Klingenberg showed that steady-state respiration in submitochondrial particles from beef heart could be modelled as a simple two-enzyme system, the first causing reduction of ubiquinone and the second causing oxidation of ubiquinol, resulting in a hyperbolic relation between the observed integrated rate of electron transfer and the rate of either oxidation or reduction of CoQ (the so-called pool equation) [4]. ...
... The functional significance of a random distribution of mitochondrial respiratory complexes had been supported in the past by the kinetic analysis of Kröger and Klingenberg for the enzymes connected by CoQ [4]. Kröger and Klingenberg showed that steady-state respiration in submitochondrial particles from beef heart could be modelled as a simple two-enzyme system, the first causing reduction of ubiquinone and the second causing oxidation of ubiquinol, resulting in a hyperbolic relation between the observed integrated rate of electron transfer and the rate of either oxidation or reduction of CoQ (the so-called pool equation) [4]. ...
... In fact, the opinion that the mitochondrial respiratory chain components are organised as ordered supramolecular aggregates in the inner membrane was widely favoured (but not undisputed) until the period of the late 1970ies and early 1980ies, when several lines of evidence were accumulated which are consistent with a random organisation of OXPHOS complexes or, at best, with a dynamic aggregate state (transient assemblies of individual respiratory complexes) as proposed by Hochman et al. (21,22). Among those findings are the result that large integral proteins are able to diffuse freely and independently over a considerable distance in various mitochondrial membrane preparations (23)(24)(25)(26)(27)(28)(29) and kinetic observations like pool behaviour of ubiquinone (e.g., 30,31). Based on these results Hackenbrock and coworkers conceptualised the "Random Collision model" contradicting any solid-state organisation of OXPHOS components in the inner membrane (27,32). ...
... Interestingly, the cogitable dissociation of respiratory supercomplexes in various mitochondrial preparations has been implied by Lenaz and Genova (20) as a reasonable explanation for the kinetic evidences in line with the random collision model which have been observed in a large number of studies (e.g., 30,31). This interpretation is based on contradicting results obtained in some corresponding studies favouring rather a solid-state organisation of respiratory complexes (e.g., 127). ...
Chapter
The supramolecular organisation of the four respiratory chain complexes as well as the FOF1-ATP synthase (complex V), comprising together the oxidative phosphorylation (OXPHOS) system, has been a matter of debate since the beginning of their biochemical investigation some decades ago. The still generally accepted view found in the textbooks is the “random collision model” regarding the five oxidative phosphorylation complexes as independent entities in the mitochondrial inner membrane. Meanwhile, the experimental evidences favouring the existence of specific respiratory supercomplexes of complexes I, III and IV as well as of ATP synthase oligomers in most eukaryotic mitochondria provide a strong collection of arguments which is not less valid than that supporting the existence of individual respiratory complexes in vivo. Of particular note is the recent determination of single particle structures of OXPHOS supercomplexes from mammals, plants, algae and yeast. Although the number of studies supporting the existence of respiratory supercomplexes has been increasing at an accelerating pace, the functional significance of respiratory supercomplexes like conceivable substrate channeling is poorly understood. Besides enzymatic advantages, an emerging crucial role appears to be the assembly/stabilisation of individual complexes. In particular, the biogenesis and/or stabilisation of complex I, the largest and most complicated respiratory complex, seem to rely on the presence of other proteins (in particular complex III and/or IV), which may integrate complex I into supramolecular structures in the inner membrane. Herein a concise but broad overview about the recent advances in the research on respiratory supercomplexes and ATP synthase oligomers is presented, attempting to put the existence of oxidative phosphorylation superstructures into perspective with the overall supramolecular organisation of interrelated mitochondrial pathways and the sophisticated subcompartmentalisation of mitochondria.
... The functional significance of a random distribution of mitochondrial complexes had been supported by the kinetic analysis of Kröger and Klingenberg [30] for the enzymes connected by Coenzyme Q; they showed that steady-state respiration in submitochondrial particles from beef heart, using either NADH or succinate as electron donors, could be modelled as a simple two-enzyme system, the first causing reduction of ubiquinone and the second causing oxidation of ubiquinol. If diffusion of the quinone and quinol species is much faster than the chemical reactions of CoQ reduction and oxidation, the quinone behaves kinetically as a homogeneous pool. ...
... Kröger and Klingenberg [30,35] already noticed that 10-20% of CoQ in submitochondrial particles is not reduced by any substrate. More recently Benard et al. [46] described the existence of three different pools of CoQ in rat liver and muscle mitochondria: one pool is utilised during succinate-dependent steady-state respiration, another (approx. ...
Article
Recent experimental evidence has replaced the random diffusion model of electron transfer with a model of supramolecular organisation based upon specific interactions between individual respiratory complexes. These supercomplexes were found to be functionally relevant by flux control analysis and to confer a kinetic advantage to NAD-linked respiration (channelling). However, the Coenzyme Q pool is still required for FAD-linked oxidations and for the proper equilibrium with Coenzyme Q bound in the supercomplex. Channelling in the cytochrome c region probably also occurs but does not seem to confer a particular kinetic advantage. The supramolecular association of individual complexes strongly depends on membrane lipid amount and composition and is affected by lipid peroxidation; it also seems to be modulated by membrane potential and protein phosphorylation. Additional properties of supercomplexes are stabilisation of Complex I, as evidenced by the destabilising effect on Complex I of mutations in either Complex III or IV, and prevention of excessive generation of reactive oxygen species. The dynamic character of the supercomplexes allows their involvement in metabolic adaptations and in control of cellular signalling pathways. This article is part of a Special Issue entitled: Dynamic and ultrastructure of bioenergetic membranes and their components.
... Q is a central component of the mtETS (Crane et al 1959;Hatefi et al 1959, Mitchell 1961 and is involved in antioxidant defense (Noh et al 2013), mitophagy (Rodríguez-Hernández et al 2009), and regulation of permeability transition (Balaban et al 2005;Bentinger et al 2007;Fontaine et al 1998;Lopez-Lluch et al 2010). Several branches of the ETS converge at the Q-junction: In mammalian mitochondria, ETS-reactive Q (redox active, Qra; Kröger, Klingenberg 1973a) is reduced by electron supply from (1) Complex I (CI), (2) CII, (3) electron-transferring flavoprotein Complex, (4) mt-glycerophosphate dehydrogenase Complex, (5) dihydro-orotate dehydrogenase Complex, and from other enzyme complexes (Enriquez, Lenaz 2014;Gnaiger 2020). Qra is oxidized downstream through CIII. ...
... An ETS-reactive Q-pool Qra is distinguished from an inactive pool mtCoQia. At steady state the redox state of Qra is proportional to respiratory rate and Qra has been considered to be homogenous, characterized as Q-pool behavior (Kröger, Klingenberg 1966, 1973a, 1973bErnster et al 1969;Hackenbrock et al 1986). However, according to Gutman (1985) there is inhomogeneity of the Qra-pool with different redox states of Q at various reduction sites (Cottingham, Moore 1983). ...
Article
Full-text available
Redox states of the mitochondrial coenzyme Q pool, which reacts with the electron transfer system, reflect the balance between (1) reducing capacities of electron flow from fuel substrates converging at the Q-junction, (2) oxidative capacities downstream of Q to O2, and (3) the load on the OXPHOS system utilizing or dissipating the protonmotive force. A three-electrode sensor (Rich 1988; Moore et al 1988) was implemented into the NextGen-O2k to monitor continuously the redox state of CoQ2 added as a Q-mimetic simultaneously with O2 consumption. The Q-Module was optimized for high signal-to-noise ratio, minimum drift, and minimum oxygen diffusion. CoQ2 equilibrates in the same manner as Q at Complexes CI, CII and CIII. The CoQ2 redox state is monitored amperometrically with the working electrode, which is poised at CoQ2 redox peak potentials determined by cyclic voltammetry. The voltammogram also provides quality control of the Q-sensor and reveals chemical interferences. The CoQ2 redox state and O2 consumption were measured simultaneously in isolated mouse cardiac and brain mitochondria. CoQ2 ― and by implication mitochondrial Q ― was more oxidized when O2 flux was stimulated by coupling control: when energy demand increased from LEAK to OXPHOS and electron transfer capacities in the succinate pathway. In contrast, CoQ2 was more reduced when O2 flux was stimulated by pathway-control of electron input capacities, increasing from the NADH (N)- to succinate (S)-linked pathway which converge at the Q-junction, with CI-Q-CIII and CII-Q-CIII segments, respectively. N- and S- respiratory pathway capacities were not completely additive, compatible with partitioning of Q intermediary between the solid-state and liquid-state models of supercomplex organization. The direct proportionality of CoQ2 reduction and electron input capacities through the CI-Q-CIII and CII-Q-CIII segments suggests that CoQ2 is accurately mimicking mitochondrial Q-redox changes.
... In humans, the most common ubiquinone contains a chain built from 10 isoprenoid units and the molecule is therefore referred to as ubiquinone-10 (Q10) or Coenzyme Q10. Q10 is well known to mediate electron and proton transport through the mitochondrial membrane for energy conversion purposes [4,5]. In its reduced form, ubiquinol, the molecule also scavenges free radicals and thereby protects the lipids from oxidation [6,7]. ...
Article
Full-text available
Ubiquinone-10 (Q10) plays a pivotal role as electron-carrier in the mitochondrial respiratory chain, and is also well known for its powerful antioxidant properties. Recent findings suggest moreover that Q10 could have an important membrane stabilizing function. In line with this, we showed in a previous study that Q10 decreases the permeability to carboxyfluorescein (CF) and increases the mechanical strength of 1-palmitoyl-2-oleyl-sn-glycero-phosphocholine (POPC) membranes. In the current study we report on the effects exerted by Q10 in membranes having a more complex lipid composition designed to mimic that of the inner mitochondrial membrane (IMM). Results from DPH fluorescence anisotropy and permeability measurements, as well as investigations probing the interaction of liposomes with silica surfaces, corroborate a membrane stabilizing effect of Q10 also in the IMM-mimicking membranes. Comparative investigations examining the effect of Q10 and the polyisoprenoid alcohol solanesol on the IMM model and on membranes composed of individual IMM components suggest, moreover, that Q10 improves the membrane barrier properties via different mechanisms depending on the lipid composition of the membrane. Thus, whereas Q10's inhibitory effect on CF release from pure POPC membranes appears to be directly and solely related to Q10's lipid ordering and condensing effect, a mechanism linked to Q10's ability to amplify intrinsic curvature elastic stress dominates in case of membranes containing high proportions of palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE).
... supercomplex | respirasome | mitochondria | respiratory chain | ubiquinone C ontemporary models for the organization of respiratorychain complexes in energy-transducing membranes range from the fluid model to the respirasome model. In the fluid model each complex is a free and independent entity, and substrates exchange freely between enzymes through common ubiquinone/ubiquinol (Q) and cytochrome c ox /c red (cyt c) pools (1)(2)(3). In the respirasome model the complexes are assembled into units that contain everything required for catalysis; substrates are channeled between enzymes within the respirasome, not exchanged with external pools (4,5). ...
Article
Full-text available
In mitochondria, four respiratory-chain complexes drive oxidative phosphorylation by sustaining a proton-motive force across the inner membrane that is used to synthesize ATP. The question of how the densely packed proteins of the inner membrane are organized to optimize structure and function has returned to prominence with the characterization of respiratory-chain supercomplexes. Supercomplexes are increasingly accepted structural entities, but their functional and catalytic advantages are disputed. Notably, substrate "channeling" between the enzymes in supercomplexes has been proposed to confer a kinetic advantage, relative to the rate provided by a freely accessible, common substrate pool. Here, we focus on the mitochondrial ubiquinone/ubiquinol pool. We formulate and test three conceptually simple predictions of the behavior of the mammalian respiratory chain that depend on whether channeling in supercomplexes is kinetically important, and on whether the ubiquinone pool is partitioned between pathways. Our spectroscopic and kinetic experiments demonstrate how the metabolic pathways for NADH and succinate oxidation communicate and catalyze via a single, universally accessible ubiquinone/ubiquinol pool that is not partitioned or channeled. We reevaluate the major piece of contrary evidence from flux control analysis and find that the conclusion of substrate channeling arises from the particular behavior of a single inhibitor; we explain why different inhibitors behave differently and show that a robust flux control analysis provides no evidence for channeling. Finally, we discuss how the formation of respiratory-chain supercomplexes may confer alternative advantages on energy-converting membranes.
... Due to their photosynthetic living, microalgae are exposed to oxidative stress and therefore need to produce antioxidative compounds. Molecular oxygen is essential for aerobic organisms because this molecule acts as an electron acceptor during mitochondria electron transport (Berg et al. 2003;Knook and Planta 1971;Kröger and Klingenberg 1973). In case of an uncompleted reduction of oxygen or due to electron leakage, reactive oxygen species (ROS) such as superoxide anions or peroxides arise. ...
Article
Full-text available
Antioxidants are substances that have the ability to reduce free, energized radicals. Thus, they prevent the oxidation of sensitive metabolites like lipids or amino acids and shield them from being destroyed by interrupting auto- or photooxidative chain reactions inside the cell. Antioxidants are also of industrial importance because they can be used as food, drug, or plastics additives. Ubiquinol, the reduced form of coenzyme Q10, is one of the most effective antioxidants in human cells. This paper explores optimization strategies to increase Q10 concentration in the biomass of Porphyridium purpureum, based on the variation of photosynthetic photon flux density. In addition, a cultivation process was performed in the 120-L scale followed by an automized extraction procedure (Accelerated Solvent Extraction®) resulting in an increase of the product recovery by a factor of 14 compared to the standard extraction method, hence reaching a specific coenzyme Q10 concentration of 141 μg g dry weight−1 and a volumetric coenzyme Q10 concentration of 1.96 mg L−1, respectively.
... Within a membrane such as the mitochondrial inner membrane, a " pool " of quinones such as ubiquinone (Q) or menaquinone (MK) diffuses in this phase, and interacts with specific quinone binding regions of the membrane protein complexes [23]. These quinones have long prenyl chains, and are virtually insoluble in water. ...
Article
Full-text available
The conventional assay method for the majority of enzymes envisages a reaction between substrates in aqueous solution. A measurable con- centration of product is accumulated over time. This paradigm has served well for the characterization of many enzymes. Variations of the method, often using chromogenic or fluorogenic substrates, have been developed and are widely used for purposes such as clinical diagnosis and screening. There are some metabolically important en- zymes for which the only published assay methods use artificial sub- strates. Some of these are oxidoreductases that use artificial mediators, and are listed in the EC list under EC 1.x.99. For computational reconstruction of the metabolism of a cell, however, it is necessary to use kinetic data from assays that reflect the physiological function in the cell, and the physiological substrates. For some oxidoreductases it is known, or considered likely that the acceptors are water-insoluble membrane-bound quinones such as ubiquinone or menaquinone, which present particular problems for measurement of kinetic para- meters. Succinate dehydrogenase/fumarate reductase is considered as an example. The oxidoreductases from membranes must be rendered soluble by detergents, which alter their kinetic behaviour. Uncertainty about the way of measuring activity of such enzymes has led to confusion in textbooks and metabolic maps, such as the persistent myth that free FAD is the acceptor for succinate dehydrogenase and related enzymes. New strategies are discussed to measure electron- transfer flux, under conditions that reflect the physiological activity of
... Dsdh2 behaves in a manner similar to wt, but Dsdh1 appears to maintain a negative midpoint redox potential and respires all available dissolved oxygen without allowing it to build up in the vessel (Figure S8). The above behavior is consistent with previous reports that respiratory rate can be directly controlled with first-order kinetics by the degree of reduction of the quinone pool in membrane vesicles and mitochondria [27,28]. We took advantage of the relatively low midpoint redox potential of menaquinone [29], and sought evidence that the respiratory rate of intact mycobacterial cells could be stimulated using the membrane permeable reducing agent dithiothreitol (DTT). ...
Article
Full-text available
In chronic infection, Mycobacterium tuberculosis bacilli are thought to enter a metabolic program that provides sufficient energy for maintenance of the protonmotive force, but is insufficient to meet the demands of cellular growth. We sought to understand this metabolic downshift genetically by targeting succinate dehydrogenase, the enzyme which couples the growth processes controlled by the TCA cycle with the energy production resulting from the electron transport chain. M. tuberculosis contains two operons which are predicted to encode succinate dehydrogenase enzymes (sdh-1 and sdh-2); we found that deletion of Sdh1 contributes to an inability to survive long term stationary phase. Stable isotope labeling and mass spectrometry revealed that Sdh1 functions as a succinate dehydrogenase during aerobic growth, and that Sdh2 is dispensable for this catalysis, but partially overlapping activities ensure that the loss of one enzyme can incompletely compensate for loss of the other. Deletion of Sdh1 disturbs the rate of respiration via the mycobacterial electron transport chain, resulting in an increased proportion of reduced electron carrier (menaquinol) which leads to increased oxygen consumption. The loss of respiratory control leads to an inability to recover from stationary phase. We propose a model in which succinate dehydrogenase is a governor of cellular respiration in the adaptation to low oxygen environments.
... Coenzyme Q10 (CoQ10), 4 also called ubiquinone, is an essential part of the mitochondrial respiratory chain that transforms energy in the diet to ATP for cellular functions (1 ). In this process of oxidative phosphorylation (OXPHOS), CoQ10 transfers reducing equivalents from respiratory complexes I and II to complex III (2 ). ...
Article
Full-text available
Background: Coenzyme Q10 (CoQ10) is an essential part of the mitochondrial respiratory chain. Unlike most other respiratory chain disorders, CoQ10 deficiency is potentially treatable. We aimed to develop and validate an accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of mitochondrial CoQ10 in clinical samples. Methods: We used mitochondria isolated from muscle biopsies of patients (n = 166) suspected to have oxidative phosphorylation deficiency. We also used fibroblast mitochondria from 1 patient with CoQ10 deficiency and 3 healthy individuals. Samples were spiked with nonphysiologic CoQ10-[(2)H6] internal standard, extracted with 1-propanol and with ethanol and hexane (2 mL/5 mL), and CoQ10 quantified by LC-MS/MS. The method and sample stability were validated. A reference interval was established from the patient data. Results: The method had a limit of quantification of 0.5 nmol/L. The assay range was 0.5-1000 nmol/L and the CVs were 7.5%-8.2%. CoQ10 was stable in concentrated mitochondrial suspensions. In isolated mitochondria, the mean ratio of CoQ10 to citrate synthase (CS) activity (CoQ10/CS) was 1.7 nmol/U (95% CI, 1.6-1.7 nmol/U). We suggest a CoQ10/CS reference interval of 1.1-2.8 nmol/U for both sexes and all ages. The CoQ10/CS ratio was 5-fold decreased in fibroblast mitochondria from a patient with known CoQ10 deficiency due to recessive prenyl (decaprenyl) diphosphate synthase, subunit 2 (PDSS2) mutations. Conclusions: Normalization of mitochondrial CoQ10 concentration against citrate synthase activity is likely to reflect most accurately the CoQ10 content available for the respiratory chain. Our assay and the established reference range should facilitate the diagnosis of respiratory chain disorders and treatment of patients with CoQ10 deficiency.
... Previous studies have addressed the effects of inhibitors [2] and mutations in and around the substrate binding site, [3,4] and made use of spectroscopy to search for ubiqsemiquinone intermediates. [5] However, they have relied on either Q 10 in native membranes [5,6] (which contain many different enzymes, thus complicating spectroscopic and kinetic analyses) or on non-physiological hydrophilic Q 10 analogues such as ubiquinone-1, ubiquinone-2 and decylubiquinone (DQ) [4,7] (which must be added in excessive concentrations to maintain steady-state catalysis and that react adventitiously at the flavin to generate damaging reactive oxygen and semiquinone species [8] ). ...
Article
Complex I is a crucial respiratory enzyme that conserves the energy from NADH oxidation by ubiquinone-10 (Q10 ) in proton transport across a membrane. Studies of its energy transduction mechanism are hindered by the extreme hydrophobicity of Q10 , and they have so far relied on native membranes with many components or on hydrophilic Q10 analogues that partition into membranes and undergo side reactions. Herein, we present a self-assembled system without these limitations: proteoliposomes containing mammalian complex I, Q10 , and a quinol oxidase (the alternative oxidase, AOX) to recycle Q10 H2 to Q10 . AOX is present in excess, so complex I is completely rate determining and the Q10 pool is kept oxidized under steady-state catalysis. The system was used to measure a fully-defined KM value for Q10 . The strategy is suitable for any enzyme with a hydrophobic quinone/quinol substrate, and could be used to characterize hydrophobic inhibitors with potential applications as pharmaceuticals, pesticides, or fungicides.
... These results are more consistent with the free exchange of Q between the membrane pool and SCs. A single pool of Q has also been demonstrated by measuring the diffusion constant of Q in the inner mitochondrial membrane 97 and the kinetics of the ubiquinone redox reactions 98,99 . Similarly, it has been shown that SC formation in Saccharomyces cerevisiae, which contains a SC III 2 +IV 2 (refs. ...
Article
The oxidative phosphorylation electron transport chain (OXPHOS-ETC) of the inner mitochondrial membrane is composed of five large protein complexes, named CI–CV. These complexes convert energy from the food we eat into ATP, a small molecule used to power a multitude of essential reactions throughout the cell. OXPHOS-ETC complexes are organized into supercomplexes (SCs) of defined stoichiometry: CI forms a supercomplex with CIII2 and CIV (SC I+III2+IV, known as the respirasome), as well as with CIII2 alone (SC I+III2). CIII2 forms a supercomplex with CIV (SC III2+IV) and CV forms dimers (CV2). Recent cryo-EM studies have revealed the structures of SC I+III2+IV and SC I+III2. Furthermore, recent work has shed light on the assembly and function of the SCs. Here we review and compare these recent studies and discuss how they have advanced our understanding of mitochondrial electron transport.
... 7,8 The CoQ10 has been reported to modulate the PTP, a mitochondrial inner membrane conductance channel, being a potential mitochondrial inhibitor of apoptotic signal transduction. 9,10 Numerous studies reported the efficacy of CoQ10 implementation in the alleviation of mitochondrial dysfunctions, such as myopathies, metabolic diseases, aging, and cardiovascular and neurodegenerative diseases. 8,11,12 In ophthalmology, the use of CoQ10 after iatrogenic damage has been supported by its protecting role against harmful free radicals after refractive surgery. ...
Article
Purpose: We evaluated the potential protective effects of Coenzyme Q10 (CoQ10) on human corneal cells and rabbit eyes after ultraviolet B (UVB) exposure and a model of wound healing in rabbit eyes after corneal epithelium removal. Methods: Human corneal epithelium cells (HCE) were exposed to a source of UVB radiation (312 nM) in the presence of different CoQ10 concentrations or vehicle. The mitochondrial function and cell survival were evaluated by means of 3-(4,5-dimethylthiazole-2-yl)2,5-diphenyl-tetrazolium (MTT) reduction and lactic dehydrogenase (LDH) release. Furthermore, quantitation of oxygen consumption and mitochondrial membrane potential were conducted. In vivo rabbit models were adopted to evaluate the effect of CoQ10 on UVB-induced conjunctival vessel hyperemia and corneal recovery after ethanol induced corneal lesion. Results: In UVB-exposed HCE cells, CoQ10 addition led to an increased survival rate and mitochondrial function. Furthermore, oxygen consumption was maintained at control levels and adenosine triphosphate (ATP) decline was completely prevented in the CoQ10-treated cells. Interestingly, in an in vivo model, CoQ10 was able dose-dependently to reduce UVB-induced vessel hyperemia. Finally, in a model of corneal epithelium removal, 12 hours from surgery, animals treated with CoQ10 showed a reduction of damaged area in respect to vehicle controls, which lasted until 48 hours. Conclusions: We demonstrated that CoQ10 reduces corneal damages after UVB exposure in vivo and in vitro by preserving mitochondrial function. Also, for the first time to our knowledge we showed that the administration of CoQ10 after corneal epithelium removal promotes corneal wound healing.
... Complex I remains partially active during acute anoxia Mindful that CI oxidizes one molecule of NADH to NAD + concomitantly reducing UQ to UQH2 in an equimolar manner while pumping four protons out of the matrix as shown in figure 1A, Jin and Bethke derived a model for CI activity using non-equilibrium thermodynamics (Jin and Bethke, 2002). We used their rate equation to generate a 3D plot of CI activity (expressed in nmol e -•min -1 •mg -1 ) and input very wide ranges of UQH2/UQ and NAD + /NADH ratio reported in the literature (Turunen et al., 2004), (Galinier et al., 2004), (Yamamoto and Yamashita, 1997), (Kroger and Klingenberg, 1973), (Kulkarni and Brookes, 2019). As shown in figure 1B, when UQH2/UQ is very high (10-20) and NAD + /NADH very low (< 1) mimicking anoxic conditions, CI activity is predicted to retain ~10% of its theoretical maximum. ...
Preprint
Full-text available
Anoxia halts oxidative phosphorylation (OXPHOS) causing hyper-reduction of mitochondrial matrix redox compounds which impedes dehydrogenases. By simultaneously measuring oxygen concentration, NADH autofluorescence, mitochondrial membrane potential and ubiquinone redox state in organello in real-time, we show that Complex I utilized endogenous quinones to oxidize NADH under acute anoxia. Untargeted or [U- ¹³ C]glutamate-targeted metabolomic analysis of matrix and effluxed metabolites extracted during anoxia and in the presence of site-specific inhibitors of the electron transfer system inferred that NAD ⁺ arising from Complex I is reduced by the oxoglutarate dehydrogenase complex yielding succinyl-CoA supporting mitochondrial substrate-level phosphorylation (mtSLP), releasing succinate. Yet, targeted metabolomic analysis using [U- ¹³ C]malate also revealed concomitant succinate dehydrogenase reversal during anoxia yielding succinate by reducing fumarate, albeit to a small extent. Our results highlight the importance of quinone availability to Complex I oxidizing NADH, thus maintaining glutamate catabolism and mtSLP in the absence of OXPHOS.
... This is not far-fetched, considering the model published by Jin and Bethke (Jin and Bethke, 2002) Acknowledging that in the reaction catalyzed by complex I one molecule of NADH is oxidized to NAD + , one molecule of Q is reduced to QH 2 and 4 protons are pumped out of the matrix, complex I activity can be expressed as a function of QH 2 /Q and NAD + /NADH. The redox ratio of CoQ9 and CoQ10 (QH 2 /Q) varies from 0.1 to 100 (Turunen et al., 2004), (Galinier et al., 2004), in plasma~20 (Yamamoto and Yamashita, 1997), and in submitochondrial particles 0.1-5 (Kroger and Klingenberg, 1973), while matrix NAD + /NADH fluctuates between 0.1 and 10 ( Kulkarni and Brookes, 2019). As shown in Fig. 4, the blue area of the 3D plot represents complex I activity (expressed in nmol*e -/min/ mg) during a wide range of QH 2 /Q and NAD + /NADH values. ...
Article
Full-text available
It is a textbook definition that in the absence of oxygen or inhibition of the mitochondrial respiratory chain by pharmacologic or genetic means, hyper-reduction of the matrix pyridine nucleotide pool ensues due to impairment of complex I oxidizing NADH, leading to reductive stress. However, even under these conditions, the ketoglutarate dehydrogenase complex (KGDHC) is known to provide succinyl-CoA to succinyl-CoA ligase, thus supporting mitochondrial substrate-level phosphorylation (mSLP). Mindful that KGDHC is dependent on provision of NAD+, hereby sources of acute NADH oxidation are reviewed, namely i) mitochondrial diaphorases, ii) reversal of mitochondrial malate dehydrogenase, iii) reversal of the mitochondrial isocitrate dehydrogenase as it occurs under acidic conditions, iv) residual complex I activity and v) reverse operation of the malate-aspartate shuttle. The concept of NAD+ import through the inner mitochondrial membrane as well as artificial means of manipulating matrix NAD+/NADH are also discussed. Understanding the above mechanisms providing NAD+ to KGDHC thus supporting mSLP may assist in dampening mitochondrial dysfunction underlying neurological disorders encompassing impairment of the electron transport chain.
... Surprisingly, information regarding the QH 2 /Q ratios during "normal" mitochondrial respiration is scant. In pioneering studies, it was found to be (somewhat) below 1 in uncoupled submitochondrial particles (i.e., the majority of the co-enzyme is oxidized [53] ). Other studies have QH 2 /Q ratios clearly above 1; see ref. [54] and references therein. ...
Article
Full-text available
Aspects of peroxisome evolution, uncoupling, carnitine shuttles, supercomplex formation, and missing neuronal fatty acid oxidation (FAO) are linked to reactive oxygen species (ROS) formation in respiratory chains. Oxidation of substrates with high FADH2/NADH (F/N) ratios (e.g., FAs) initiate ROS formation in Complex I due to insufficient availability of its electron acceptor (Q) and reverse electron transport from QH2, e.g., during FAO or glycerol‐3‐phosphate shuttle use. Here it is proposed that the Q‐cycle of Complex III contributes to enhanced ROS formation going from low F/N ratio substrates (glucose) to high F/N substrates. This contribution is twofold: 1) Complex III uses Q as substrate, thus also competing with Complex I; 2) Complex III itself will produce more ROS under these conditions. I link this scenario to the universally observed Complex III dimerization. The Q‐cycle of Complex III thus again illustrates the tension between efficient ATP generation and endogenous ROS formation. This model can explain recent findings concerning succinate and ROS‐induced uncoupling.
... In this updated view, part of mitochondrial CoQ10 is bound within supercomplexes where electron transfer would be mediated by tunneling or microdiffusion, with a clear kinetic advantage. CoQ10 content in mitochondria is in large excess compared to the prosthetic groups of respiratory enzymes [7] and the unbound mitochondria CoQ10 pool, besides constituting a reservoir for bioenergetic requirements and an antioxidant for mitochondrial membrane lipids, is able to modulate bioenergetic processes and cell death pathways by binding to specific proteins, such as uncoupling proteins (UCPs) [8] and the permeability transition pore (PTP) [9]. ...
Article
Full-text available
Coenzyme Q10 (CoQ10) is an endogenous lipophilic quinone, ubiquitous in biological membranes and endowed with antioxidant and bioenergetic properties, both crucial to the aging process. In fact, coenzyme Q10 synthesis is known to decrease with age in different tissues including skin. Moreover, synthesis can be inhibited by 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase inhibitors such as statins, that are widely used hypocholesterolemic drugs. They target a key enzymatic step along the mevalonate pathway, involved in the synthesis of both cholesterol and isoprenylated compounds including CoQ10.In the present study, we show that pharmacological CoQ10 deprivation at concentrations of statins > 10000 nM triggers intracellular oxidative stress, mitochondrial dysfunction and generates cell death in human dermal fibroblasts (HDF). On the contrary, at lower statin concentrations, cells and mainly mitochondria, are able to partially adapt and prevent oxidative imbalance and overt mitochondrial toxicity. Importantly, our data demonstrate that CoQ10 decrease promotes mitochondrial permeability transition and bioenergetic dysfunction leading to premature aging of human dermal fibroblasts in vitro.
... The photosynthetic and the respiratory apparatuses of facultative phototrophic bacteria contain several supra-molecular m em brane bound complexes, namely the reaction center and light-harvesting complexes, the ubiquinol-cytochrom e b /c oxidoreductase, the cytochrome oxidase and the coupling factor [1,2]. The functional interaction of these complexes may proceed through direct close contact between the redox partners or through mobile carriers which diffuse laterally in the lipid double layer [3][4][5][6]. The observed oxidation half-tim es are quite different: 5 ms for cytochrome c oxidase, 80 ms for the cytochromes b and 500 ms for the dehydrogenases [7], Because of a high p ro tein/lipid ratio in the electron transport m em branes and strong protein-protein as well as p ro tein/lipid inter actions the mobility of single m em brane com po nents is lower in electron transport m em branes than in other membranes [8]. ...
Article
Full-text available
Intracytoplasmic membrane vesicles (chromatophores) isolated from Rhodopseudomonas capsulata cells were fused with liposomes by a pH transition procedure. Vesicles of lower density and higher lipid contents and larger diameter than chromatophores were obtained. Similar results were observed by Ca2+ induced fusion and by the freeze-thawing method. Respiratory and light-induced electron transport were measured in chromatophores and fused vesicles. Light-induced reaction center bleaching was observed in all types of vesicles, whereas repiratory electron transport was substantially diminished by lipid incorporation. Ubiquinone 10 restored to some extent respiratory electron transport and oxidative phosphorylation and it modified the photophosphorylation kinetics under continuous light. Electrochromic carotenoid band-shift and the 9-aminoacridine fluorescence quenching indicate that the capacity of the fused vesicles to maintain an electrochemical proton gradient has not been substantially diminished. From the kinetics of 9-aminoacridine quenching an increased K+-permeability seems to be apparent.
... Whereas in the fluid model, individual ETC complexes and redox components move in diffusional motion constantly and independently in the membrane, and the electrons are transferred between the complexes through the free diffusion of Q and cyt c (Hackenbrock, 1977). The fluid model gained general recognition for decades and was consistent with the chemiosmotic hypothesis (Hatefi et al., 1962;Kroger and Klingenberg, 1973;Hochli and Hackenbrock, 1976;Hochli et al., 1985;Gupte and Hackenbrock, 1988). However, as time went on, neither of these two models could withstand all of the surfacing experimental evidence. ...
Article
Full-text available
Respirasome, as a vital part of the oxidative phosphorylation system, undertakes the task of transferring electrons from the electron donors to oxygen and produces a proton concentration gradient across the inner mitochondrial membrane through the coupled translocation of protons. Copious research has been carried out on this lynchpin of respiration. From the discovery of individual respiratory complexes to the report of the high-resolution structure of mammalian respiratory supercomplex I1III2IV1, scientists have gradually uncovered the mysterious veil of the electron transport chain (ETC). With the discovery of the mammalian respiratory mega complex I2III2IV2, a new perspective emerges in the research field of the ETC. Behind these advances glitters the light of the revolution in both theory and technology. Here, we give a short review about how scientists 'see' the structure and the mechanism of respirasome from the macroscopic scale to the atomic scale during the past decades.
Chapter
In this chapter we provide a review with a focus on the function of Coenzyme Q (CoQ, ubiquinone) in mitochondria. The notion of a mobile pool of CoQ in the lipid bilayer as the vehicle of electrons from respiratory complexes has somewhat changed with the discovery of respiratory supramolecular units, in particular the supercomplex comprising Complexes I and III; in such assembly the electron transfer is thought to be mediated by direct channelling, and we provide evidence for a kinetic advantage on the transfer based on random collisions. The CoQ pool, however, has a fundamental function in establishing a dissociation equilibrium with bound CoQ, besides being required for electron transfer from other dehydrogenases to Complex III. CoQ bound to Complex I and to Complex III is also involved in proton translocation; although the mechanism of the Q-cycle is well established for Complex III, the involvement of CoQ in proton translocation by Complex I is still debated. This review also briefly examines some additional roles of CoQ, such as the antioxidant effect of its reduced form and its postulated action at the transcriptional level.
Article
Full-text available
Aims: The mitochondrial respiratory chain is recognized today to be arranged in supramolecular assemblies (supercomplexes). Besides conferring a kinetic advantage (substrate channeling) and being required for the assembly and stability of Complex I, indirect considerations support the view that supercomplexes may also prevent excessive formation of reactive oxygen species (ROS) from the respiratory chain. In the present study, we have directly addressed this issue by testing the ROS generation by Complex I in two experimental systems in which the supramolecular organization of the respiratory assemblies is impaired by: (i) treatment either of bovine heart mitochondria or liposome-reconstituted supercomplex I-III with dodecyl maltoside; (ii) reconstitution of Complexes I and III at high phospholipids to protein ratio. Results: The results of our investigation provide experimental evidence that the production of ROS is strongly increased in either model, supporting the view that disruption or prevention of the association between Complex I and Complex III by different means enhances the generation of superoxide from Complex I. Innovation: Dissociation of supercomplexes may link oxidative stress and energy failure in a vicious circle. Conclusion: Our findings support a central role of mitochondrial supramolecular structure in the development of the aging process and in the etiology and pathogenesis of most major chronic diseases.
Article
Full-text available
Coenzyme Q (CoQ) is a key component of the respiratory chain of all eukaryotic cells. Its function is closely related to mitochondrial respiration, where it acts as an electron transporter. However, the cellular functions of coenzyme Q are multiple: it is present in all cell membranes, limiting the toxic effect of free radicals, it is a component of LDL, it is involved in the aging process, and its deficiency is linked to several diseases. Recently, it has been proposed that coenzyme Q contributes to suppressing ferroptosis, a type of iron-dependent programmed cell death characterized by lipid peroxidation. In this review, we report the latest hypotheses and theories analyzing the multiple functions of coenzyme Q. The complete knowledge of the various cellular CoQ functions is essential to provide a rational basis for its possible therapeutic use, not only in diseases characterized by primary CoQ deficiency, but also in large number of diseases in which its secondary deficiency has been found.
Article
A kinetic study of the effect of antimycin A on succinate oxidase from plant mitochondria produced sigmoidal curves for the reduction of cytochromes b560 and b585 and for the inhibition of succinate oxidase. In the stationary state the interaction of the various components of the respiratory chain (flavins, ubiquinone, cytochromes…) occurs in a sequential mode which allows the application of simple equations utilizing rate constants and cytochrome concentrations. In these equations, it is assumed that there exists an excess of ubiquinone relative to other components of the respiratory chain as suggested by Kröger & Klingenberg (1970) and that the reoxidation of b cytochromes is fast. The inhibition by antimycin A, characterized by non-linear inhibition curves for succin-oxidase and inhibitor fixation in complex III on a component other than cytochrome c1, is interpreted in terms of this model. This hypothesis presupposes the existence of an F factor between cytochrome b560 and cytochrome c1 as suggested by other authors. Utilizing these equations, theoretical curves for the inhibition of the reduction of cytochrome b560 have been constructed and the results agree with the experimental data. The kinetic behavior of cytochrome b566 during the induction of anaerobiosis suggests that it is not directly involved in the electron transfer chain but rather is either in thermodynamic equilibrium with cytochrome b560 or in a shunt between cytochrome b560 and factor F. From the experimental data, an equation is derived for the inhibition of the reduction of cytochrome b566 by antimycin A. The actual effects of ATP and mClCCP on succinoxidase agree well with those predicted by the model.
Article
Full-text available
Crystal structure of trans-atovaquone (antimalarial drug), its polymorph and its stereoisomer (cis) along with five other derivatives with different functional groups have been analyzed. Based on the conformational features of these compounds and the characteristics of the nature of intermolecular interactions, valuable insights into the atomistic details of protein–inhibitor interactions have been derived by docking studies. Atovaquone and its derivatives pack in the crystal lattice using intermolecular O–H⋯O hydrogen bond dimer motifs supported by surrogate weak interactions including C–H⋯O and C–H⋯Cl hydrogen bonds. The docking results of these molecules with cytochrome bc1 show preferences to form N–H⋯O, O–H⋯O and O–H⋯Cl hydrogen bonds. The involvement of halogen atoms in the binding pocket appears to be significant and is contrary to the theoretically predicted mechanism of protein–ligand docking reported earlier based on mimicking experimental binding results of stigmatellin with cytochrome bc1. The significance of subtle energy factors controlled by weak intermolecular interactions appears to play a major role in drug binding. For details: https://doi.org/10.1039/C3CE40336J
Article
. Coenzyme Q10 (CoQ10) and citrate synthase (CS) activities were analysed in the myocardium of brain-dead organ donors (14–40 years). Different parts of the heart were studied: right and left auricular appendage, right and left atrium, right ventricle (septum and free wall) and left ventricle (septum, free wall, and papillary muscle). Freeze-dried, dissected myocardial samples were analysed for CoQ10 content by HPLC and CS activity by fluorometric technique. CoQ10 content in the normal human myocardium was lowest in auricular appendages and atria (0·25 ±0·06 mg×g-1 dry muscle), intermediate in right ventricle (0·37±0·05 mg×g-1 dm) and highest in left ventricle (0·42±0·07 mg×g-1 dm). CS activity showed the same relationship between these locations as CoQ10. The results suggest that there exist differences in CoQ10 content between different parts of the normal human myocardium. These differences were closely related to the differences in CS activity between corresponding parts. The differences between different parts of the heart may be related to divergent work demand, and the constant relationship between CS and CoQ10 may be related to their coupling to the mitochondrial oxidative metabolism.
Article
Antimycin and cyazofamid are specific inhibitors of the mitochondrial respiratory chain, and bind to the Qi site of the cytochrome bc1 complex. With the aim to understand the detailed molecular inhibition mechanism of Qi inhibitors, we performed a comparative investigation of the inhibitory kinetics of them against the porcine bc1 complex. The results showed that antimycin is a slow tight-binding inhibitor of succinate-cytochrome c reductase with $${K_i}$$ = 0.033 ± 0.00027 nM and noncompetitive inhibition with respect to cytochrome c. Cyazofamid is a classical inhibitor of succinate-cytochrome c reductase with $${K_i}$$= 12.90 ± 0.91 μM and a noncompetitive inhibitor with respect to cytochrome c. Both of them show competitive inhibition with respect to substrate DBH2 . Further molecular docking and quantum mechanics calculations were performed. The results showed that antimycin underwent significant conformational change upon the binding. The energy barrier between the conformations in the crystal and in the binding pocket is ~13.63 kcal/mol. Antimycin formed a H-bond with Asp228 and two water-bridged H-bonds with Lys227 and His201, whereas cyazofamid formed only one H-bond with Asp228. The conformational change and the different hydrogen bonding network might account for why antimycin is a slow-tight inhibitor, whereas cyazofamid is a classic inhibitor. This article is protected by copyright. All rights reserved.
Chapter
Coenzyme Q10 (CoQ10) is an essential part of the mitochondrial respiratory chain. Here, we describe an accurate and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of mitochondrial CoQ10 in isolated mitochondria. In the assay, mitochondrial suspensions are spiked with CoQ10-[²H9] internal standard (IS), extracted with organic solvents and CoQ10 quantified by LC-MS/MS using multiple reaction monitoring (MRM).
Article
Full-text available
Under aerobic conditions, mitochondrial oxidative phosphorylation (OXPHOS) converts the energy released by nutrient oxidation into ATP, the currency of living organisms. The whole biochemical machinery is hosted by the inner mitochondrial membrane (mtIM) where the protonmotive force built by respiratory complexes, dynamically assembled as super-complexes, allows the F1FO-ATP synthase to make ATP from ADP + Pi. Recently mitochondria emerged not only as cell powerhouses, but also as signaling hubs by way of reactive oxygen species (ROS) production. However, when ROS removal systems and/or OXPHOS constituents are defective, the physiological ROS generation can cause ROS imbalance and oxidative stress, which in turn damages cell components. Moreover, the morphology of mitochondria rules cell fate and the formation of the mitochondrial permeability transition pore in the mtIM, which, most likely with the F1FO-ATP synthase contribution, permeabilizes mitochondria and leads to cell death. As the multiple mitochondrial functions are mutually interconnected, changes in protein composition by mutations or in supercomplex assembly and/or in membrane structures often generate a dysfunctional cascade and lead to life-incompatible diseases or severe syndromes. The known structural/functional changes in mitochondrial proteins and structures, which impact mitochondrial bioenergetics because of an impaired or defective energy transduction system, here reviewed, constitute the main biochemical damage in a variety of genetic and age-related diseases.
Article
Full-text available
The gut microbiome is shaped by diet and influences host metabolism; however, these links are complex and can be unique to each individual. We performed deep metagenomic sequencing of 1,203 gut microbiomes from 1,098 individuals enrolled in the Personalised Responses to Dietary Composition Trial (PREDICT 1) study, whose detailed long-term diet information, as well as hundreds of fasting and same-meal postprandial cardiometabolic blood marker measurements were available. We found many significant associations between microbes and specific nutrients, foods, food groups and general dietary indices, which were driven especially by the presence and diversity of healthy and plant-based foods. Microbial biomarkers of obesity were reproducible across external publicly available cohorts and in agreement with circulating blood metabolites that are indicators of cardiovascular disease risk. While some microbes, such as Prevotella copri and Blastocystis spp., were indicators of favorable postprandial glucose metabolism, overall microbiome composition was predictive for a large panel of cardiometabolic blood markers including fasting and postprandial glycemic, lipemic and inflammatory indices. The panel of intestinal species associated with healthy dietary habits overlapped with those associated with favorable cardiometabolic and postprandial markers, indicating that our large-scale resource can potentially stratify the gut microbiome into generalizable health levels in individuals without clinically manifest disease. Analyses from the gut microbiome of over 1,000 individuals from the PREDICT 1 study, for which detailed long-term diet information as well as hundreds of fasting and same-meal postprandial cardiometabolic blood marker measurements are available, unveil new associations between specific gut microbes, dietary habits and cardiometabolic health.
Article
Full-text available
Mitochondrial respiratory supercomplexes, comprising complexes I, III, and IV, are the minimal functional units of the electron transport chain. Assembling the individual complexes into supercomplexes may stabilize them, provide greater spatiotemporal control of respiration, or, controversially, confer kinetic advantages through the sequestration of local quinone and cytochrome c pools (substrate channeling). Here, we have incorporated an alternative quinol oxidase (AOX) into mammalian heart mitochondrial membranes to introduce a competing pathway for quinol oxidation and test for channeling. AOX substantially increases the rate of NADH oxidation by O2 without affecting the membrane integrity, the supercomplexes, or NADH-linked oxidative phosphorylation. Therefore, the quinol generated in supercomplexes by complex I is reoxidized more rapidly outside the supercomplex by AOX than inside the supercomplex by complex III. Our results demonstrate that quinone and quinol diffuse freely in and out of supercomplexes: substrate channeling does not occur and is not required to support respiration.
Article
Full-text available
The rediscovery and reinterpretation of the Warburg effect in the year 2000 occulted for almost a decade the key functions exerted by mitochondria in cancer cells. Until recent times, the scientific community indeed focused on constitutive glycolysis as a hallmark of cancer cells, which it is not, largely ignoring the contribution of mitochondria to the malignancy of oxidative and glycolytic cancer cells, being Warburgian or merely adapted to hypoxia. In this review, we highlight that mitochondria are not only powerhouses in some cancer cells, but also dynamic regulators of life, death, proliferation, motion and stemness in other types of cancer cells. Similar to the cells that host them, mitochondria are capable to adapt to tumoral conditions, and probably to evolve to 'oncogenic mitochondria' capable of transferring malignant capacities to recipient cells. In the wider quest of metabolic modulators of cancer, treatments have already been identified targeting mitochondria in cancer cells, but the field is still in infancy.
Article
Lipid composition determines the physical properties of biological membranes and can vary substantially between and within organisms. We describe a specific role for the viscosity of energy-transducing membranes in cellular respiration. Engineering of fatty acid biosynthesis in Escherichia coli allowed us to titrate inner membrane viscosity across a 10-fold range by controlling the abundance of unsaturated or branched lipids. These fluidizing lipids tightly controlled respiratory metabolism, an effect that can be explained with a quantitative model of the Electron Transport Chain (ETC) that features diffusion-coupled reactions between enzymes and electron carriers (quinones). Lipid unsaturation also modulated mitochondrial respiration in engineered budding yeast strains. Thus, diffusion in the ETC may serve as an evolutionary constraint for lipid composition in respiratory membranes.
Article
The mitochondrial electron transport chain complexes are organized into supercomplexes (SCs) of defined stoichiometry, which have been proposed to regulate electron flux via substrate channeling. We demonstrate that CoQ trapping in the isolated SC I+III2 limits complex (C)I turnover, arguing against channeling. The SC structure, resolved at up to 3.8 Å in four distinct states, suggests that CoQ oxidation may be rate limiting because of unequal access of CoQ to the active sites of CIII2. CI shows a transition between "closed" and "open" conformations, accompanied by the striking rotation of a key transmembrane helix. Furthermore, the state of CI affects the conformational flexibility within CIII2, demonstrating crosstalk between the enzymes. CoQ was identified at only three of the four binding sites in CIII2, suggesting that interaction with CI disrupts CIII2 symmetry in a functionally relevant manner. Together, these observations indicate a more nuanced functional role for the SCs.
Chapter
Coenzyme Q10 (CoQ10) is an essential part of the mitochondrial respiratory chain. Here, we describe an accurate and sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of mitochondrial CoQ10 in isolated mitochondria. In the assay, mitochondrial suspensions are spiked with CoQ10-[2H6] internal standard, extracted with organic solvents, and CoQ10 quantifi ed by LC-MS/MS using multiple reaction monitoring (MRM).
Chapter
The metabolic capacity of the eukaryotic cell to convert free energy contained in nutrients into ATP is a process accomplished by a multi-step system: the mitochondrial respiratory chain. This chain involves a series of electron-transferring enzymes and redox co-factors, whose biochemical characterization is the collective result of more than 50 years of scientists’ endeavors. The current knowledge describes in detail the structure and function of the individual proton-translocating “core” complexes of the respiratory chain (Complex I, III, IV). However, a holistic approach to the study of electrons transport from NAD-dependent substrates to oxygen has recently directed our attention to the existence of specific albeit dynamic interactions between the respiratory complexes. In this context, the respiratory complexes are envisaged to be either in form of highly ordered assemblies (i.e. supercomplexes) or as individual enzymes randomly distributed in the mitochondrial membrane. Either model of organization has functional consequences, which can be discussed in terms of the structural stability of the protein complexes and the kinetic efficiency of inter-complex electron transfer. Available experimental evidence suggests that Complex I and Complex III behave as assembled supercomplexes (ubiquinone-channeling) or as individual enzymes (ubiquinone-pool), depending on the lipid environment of the membrane. On the contrary, a strict association of Complexes III and Complex IV is not required for electron transfer via cytochrome c, although there are supercomplexes in bovine heart mitochondria, known as the respirasomes, that also include some molecules of Complex IV. Our recent experimental results demonstrate that the disruption of the supercomplex I1–III2 enhances the propensity of Complex I to generate the superoxide anion; we propose that any primary source of oxidative stress in mitochondria may perpetuate generation of reactive oxygen species by a vicious cycle involving supercomplex dissociation as a major determinant.
Chapter
Plant respiration has long been described to be resistant to cyanide, since the first observation was made in 1929 by Genevois on sweet pea (Lathyrus odora-tus) seedlings. Soon after, Van Herk and Badenhuizen (1934) and Van Herk (1937 a, b, c) showed that the respiration of the spadix of the Sauromatum guttatum was highly resistant to cyanide. Respiration in this group of plants (Araceae) is known to be extremely high and linked to heat production, particularly during pollination (see Lance 1972, Meeuse 1975). In 1939, Okunuki observed that the respiration of Sauromatum pollen was resistant to carbon monoxide, and a similar observation was also made by Marsh and Goddard (1939) on carrot leaves. These pioneering works established the concept that plant respiration differed from that of animals by its behavior toward respiratory inhibitors. Cyanide could even stimulate respiration, as in potato tubers (Hanes and Barker 1931).
Chapter
The aging process is characteristically associated with a progressive decline in physiological functions. This is likely related to a failure of mitochondrial energy production as a consequence of impairment of the mitochondria) oxidative phosphorylation system.
Chapter
Evidence from several investigations demonstrates the existence of supramolecular units of Complex I, Complex III, and multiple copies of Complex IV in mitochondria and indicates that specific respiratory complexes may preferentially associate to form cytochrome-containing supercomplexes in the native membrane. There are now indications thatcardiolipin,a distinctive mitochondrial lipid, stabilizes the respiratory assemblies. The isolated supercomplexes are active with respect to both their component individual complexes and the entire respiratory function that relies on Coenzyme Q and cytochrome c as intermediate substrates. The latter finding argues against previous models of a random distribution of the respiratory complexes in mitochondria. The supercomplex organization is compatible with electron transfer, but experimental evidence is scant for an effective mechanism via substrate channeling compared to free diffusion of substrates in accordance with the random collision model. The finding that Complex I is almost totally associated in a supercomplex with Complex III seems to exclude a role for the ubiquinone pool in physiological electron transfer between these two complexes, whereas it is certainly required for electron transfer from Complex II or from other dehydrogenases to Complex III; likely, only a small fraction of Complex IV forms a functional supercomplex with channeling of cytochrome c. Nevertheless, the supercomplexes may physiologically exist in equilibrium with free complexes (plasticity model). The supercomplex organization appears to prevent excessive generation of reactive oxygen species from the respiratory chain; accordingly, many pathological conditions and the mitochondrial aging phenotype characterized by a loss of supercomplex assembly correlate with mitochondrial dysfunction and increased oxidative stress. Specific metabolic signals may also arise in the cell in response to a tuned production of reactive oxygen species as a consequence of the controlled dynamics of supercomplex assembling/disassembling at different physio-pathological conditions. The present review paper provides an updated and extensive discussion on the subject.
Article
Full-text available
Previous microbiome and metabolome analyses exploring non-communicable diseases have paid scant attention to major confounders of study outcomes, such as common, pre-morbid and co-morbid conditions, or polypharmacy. Here, in the context of ischemic heart disease (IHD), we used a study design that recapitulates disease initiation, escalation and response to treatment over time, mirroring a longitudinal study that would otherwise be difficult to perform given the protracted nature of IHD pathogenesis. We recruited 1,241 middle-aged Europeans, including healthy individuals, individuals with dysmetabolic morbidities (obesity and type 2 diabetes) but lacking overt IHD diagnosis and individuals with IHD at three distinct clinical stages—acute coronary syndrome, chronic IHD and IHD with heart failure—and characterized their phenome, gut metagenome and serum and urine metabolome. We found that about 75% of microbiome and metabolome features that distinguish individuals with IHD from healthy individuals after adjustment for effects of medication and lifestyle are present in individuals exhibiting dysmetabolism, suggesting that major alterations of the gut microbiome and metabolome might begin long before clinical onset of IHD. We further categorized microbiome and metabolome signatures related to prodromal dysmetabolism, specific to IHD in general or to each of its three subtypes or related to escalation or de-escalation of IHD. Discriminant analysis based on specific IHD microbiome and metabolome features could better differentiate individuals with IHD from healthy individuals or metabolically matched individuals as compared to the conventional risk markers, pointing to a pathophysiological relevance of these features.
Article
There are many similarities between the oxidative phosphorylation apparatus of mitochondria and those found in the cytoplasmic membranes of alpha‐proteobacteria, exemplified by Paracocus denitrificans. These similarities are reviewed here alongside consideration of the differences between mitochondrial and bacterial counterparts, as well as the loss from the modern mitochondria of many of the bacterial respiratory proteins. The assembly of c‐type cytochromes is of particular evolutionary interest as the post‐translational apparatus used in the alpha‐proteobacteria is found in plants, and for example in eukyarotic species including algae of various kinds together with jakobids, but has been superseded by different systems in mitochondria of metazoans and trypanosomatids. All mitochondrial cytochromes c have the N‐terminal sequence feature that is recognised by the metazoan system whereas the bacterial counterparts do not, suggesting that the loss of the bacterial system from eukaryotes occurred in the context of an already present recognition sequence in the eukaryotic cytochromes. Interestingly, in the case of cytochromes c1 the putative recognition features for the metazoans appear to be substantially present in the bacterial proteins. The ability to prepare from P. denitrificans inverted membrane vesicles with classic respiratory control presents a valuable system from which to draw lessons concerning the long debated topic of what controls the rates of respiration and ATP synthesis in mitochondria.
Book
This book focuses on the use of various molecules with antioxidant properties in the treatment of major male genital tract disorders, especially male infertility, erectile dysfunction, and accessory gland infection. The coverage also includes discussion of pathophysiology, the molecular basis of male infertility, and the rationale for use of antioxidants, with particular attention to coenzyme Q10 and carnitine. Oxidative stress occurs when the production of reactive oxygen species, including free radicals, exceeds the body’s natural antioxidant defences, leading to cellular damage. Oxidative stress is present in about half of all infertile men, and reactive oxygen species can produce infertility both by damaging the sperm membrane, with consequences for sperm motility, and by altering the sperm DNA. There is consequently a clear rationale for the use of antioxidant treatments within andrology, and various in vitro and in vivo studies have indicated that many antioxidants indeed have beneficial impacts. In providing a detailed and up-to-date overview of the subject, this book will be of interest to both practitioners and researchers in andrology, endocrinology, and urology.
Article
Abstract Substantial evidence exists that the mitochondrial respiratory chain is organized in supramolecular units called supercomplexes or respirasomes. While the structural evidence of the supercomplexes is overwhelming, fewer studies were addressed to their functional relevance. Although the presence of Coenzyme Q channelling between Complexes I and III is ascertained, no such demonstration has been clearly done for cytochrome c between Complexes III and IV, at least in mammalian mitochondria. This review also discusses the implications concerning the amount of respiratory complexes organized in supercomplexes and the possibility that they represent associations in dynamic equilibrium with the individual complexes.
Article
Full-text available
The maximum possible bimolecular rate of electron transfer to ubiquinone (coenzyme Q with six isoprenoid units in the side chain) is likely to be that from solvated electrons, now determined by pulse radiolysis to be 1.7 x 10¹⁰m⁻¹ sec⁻¹. ·CH2O⁻ radicals also reduce ubiquinone, with k = 2.0 x 10⁹m⁻¹ sec⁻¹. The reduction product in both cases is the ubisemiquinone free radical anion, ε445 nm = 7200 m⁻¹ cm⁻¹. In acid solutions reduction is produced only by ·CH2OH radicals with k = 1.4 x 10⁹m⁻¹ sec⁻¹. The reduction product is the neutral ubisemiquinone free radical, ε420 nm = 3000 m⁻¹ cm⁻¹. Neutral ubisemiquinone free radicals disproportionate with k = 4.8 x 10⁷m⁻¹ sec⁻¹. In neutral solution ubiquinone is reduced both by solvated electrons and by ·CH2OH radicals: formation of the neutral ubisemiquinone is followed by deprotonation to yield the anion, with k = 1.0 x 10⁴ sec⁻¹. The pK of the semiquinone free radical is 6.45 ± 0.15.
Article
In succinoxidase, CoQ1 had low activity, and CoQ2 through CoQ10 had comparable activities in both the acetone- and pentane-extracted systems. In the DPNH-oxidase system, CoQ1 had low activity; CoQ2, CoQ3, and CoQ4 had about one-fourth the activity; and CoQ5 and CoQ6 had about one-half the activity of CoQ10. CoQ7, CoQ8, and CoQ9 had activity comparable with that of CoQ10.Hexahydro- and octahydrocoenzyme Q4 had activities comparable with that of CoQ4 and CoQ10 for succinoxidase, but about 10–15% that of CoQ10 for DPNH-oxidase. The double bond in the isoprenoid unit adjacent to the nucleus, and possible cyclization, are not necessary for activity. 2,3-Dimethoxy-5-methyl-6-heptadecyl-l,4-benzoquinone shows succinoxidase and DPNH-oxidase activities comparable with that of hexahydrocoeuzyme Q4. The 5-methyl group is not essential for succinoxidase activity, but has some significance for DPNH-oxidase. A tertiary hydroxyl group in the isoprenoid unit adjacent to the nucleus abolishes activity for succinoxidase but not completely for DPNH-oxidase. Rhodoquinone had no activity in the succinoxidase system and 25% that of CoQ10 in the DPNH-oxidase system.Representatives of biosynthetic precursors, 2-methoxy-5-methyl-6-phytyl-1,4-benzoquinone and 2-methoxy-3-hydroxy-5-methyl-6-phytyl-1,4-benzoquinone, had no activity in either the succinoxidase or DPNH-oxidase systems. Four compounds having a mechanistic relationship between CoQ and vitamin E had no activity. These data support the concept of two sites for the electron transfer of CoQ. The site for CoQ in succinoxidase is not very specific in the structural and steric requirement for the isoprenoid side chain, and has an electron potentiality that is not met by rhodoquinone. The site for CoQ in DPNH-oxidase has a rather specific requirement for a given length of side chain, and an electron potentiality that is significantly met by rhodoquinone.
Article
The rate of reduction of ubiquinone by NADH in electron transport particles (ETP) in the absence of inhibitor, and in the presence of cyanide or Antimycin A, has been determined spectrophotometrically in a rapid-mixing stopped flow apparatus, and compared with the rate of reduction of the cytochromes under the same conditions. With 20–50 μm NADH as substrate, and inhibitor concentrations low enough to allow cycles of reduction and reoxidation, ubiquinone remains oxidized in the absence of inhibitor and the presence of cyanide, but is reduced in the presence of Antimycin A with a half-time for reduction of 0.9 second. In contrast, cytochromes, c,c1, and flavoprotein have half-times for reduction of 70–100 mseconds in the presence of cyanide; cytochrome b is reduced with a half-time of 30–40 mseconds in the presence of Antimycin A. With 100 μm NADH as substrate and 3 mm KCN as inhibitor, 74% of the ubiquinone is reduced in these particles. Cyanide has a minor, but definite inhibiting effect on the reduction of cytochrome b. These results, and those reported in two previous papers are summarized, and a scheme for electron transport in ETP is put forward.
Article
The half-time for oxidation of reduced ubiquinone in electron transport particles (ETP) made anaerobic with succinate is 3–6 msec and is comparable to that for cytochrome c which lies between 2 and 3 msec in these particles. The half-time for oxidation of reduced cytochrome b is much greater, being in the range of 60–100 msec. These results imply that ubiquinone and cytochrome b form two branches of electron transport between succinate and cytochrome c, but that most of the electron transport occurs through ubiquinone. Previous work had showed that ubiquinone is peripheral to the NADH oxidase pathway in these particles (4). Comparison of these rates in ETP with rates of electron transport through cytochrome b and ubiquinone in intact mitochondria (15) and in submitochondrial particles derived by sonication (16) indicates that increasing disruption of mitochondrial structure decreases the rate of electron flux through cytochrome b and increases the rate through ubiquinone. Assignment to ubiquinone of a central function in electron transport, based on experiments involving disruption and fractionation of the electron transport chain. would appear to be unjustified.
Article
1. The incorporation of small amounts of ubiquinone (Q) into pentane-extracted submitochondrial particles has been studied. A procedure is described which allows nearly quantitative incorporation of as little as 0.2 to 0.3 nmol Q/mg protein, i.e. less than 10% of the Q present in the lyophilized particles prior to extraction. 2. It is shown that both NADH oxidase and succinate oxidase activities can be restored to 100% of that in lyophilized particles by incorporation of 6–8 nmol Q/mg protein. The relative activities of both oxidases show a parallel response to an increase of the Q content of the particles between 0.2 and 22 nmol Q/mg protein, 50% reactivation being obtained at about 2 nmol Q/mg protein. 3. 50 to 60% of the incorporated Q can be reduced by NADH or succinate, independent of the total concentration of Q in the membrane. Similar reduction values were obtained for lyophilized particles prior to extraction, suggesting that incorporated Q functions similarly to that originally present in the particles. 4. Succinate dehydrogenase activity, which is decreased 50% by extraction of Q, can be completely restored upon incorporation of only 1.5 nmol Q/mg protein. 5. Extraction of lyophilized particles with pentane +10% acetone results in a more effective removal of Q, but also in a differential Q requirement for NADH oxidase and succinate oxidase. Thin-layer chromatography shows that extraction with pentane + acetone removes an additional, unidentified, nonpolar lipid. 6. The present data do not support the belief that succinate oxidase and NADH oxidase communicate with separate pools of Q.
Article
1. Substituted benzoquinones and napthoquinones can function as electron acceptors for the NADH dehydrogenase segment of the mitochondrial electron transport system. Anthraquinones and several hydroxylated quinones are not reduced by the enzyme complex. 2. Piericidin A treatment at concentrations known to inhibit electron transport causes varying degrees of inhibition of quinone reductase activities. The pattern of piericidin A inhibition suggests that certain quinones are reduced at sites either before or after the piericidin A inhibition site. Reduction of quinones such as 5-hydroxy-1,4-napthoquinone (juglone) and 1,2-napthoquinone is inhibited only slightly. Reduction of ubiquinone 1 and 2 and 2,3,5,6-tetramethyl-1,4-benzoquinone (duroquinone) is almost completely inhibited following piericidin A treatment. 3. Comparison of juglone reductase activity with ferricyanide reductase activity suggests that these acceptors are reduced at nonequivalent sites in the NADH dehydrogenase. Juglone reductase activity is stimulated following mercurial treatment while ferricyanide reductase activity is slightly inhibited. Preincubation of NADH dehydrogenase with NADH followed by mercurial treatment causes almost complete inhibition of ferricyanide reductase activity. Activation of juglone reductase activity still occurs under these conditions. The activation is specific for preparations of NADH dehydrogenase, for mercurials, and for napthoquinone reductase activity.
Article
Coenzyme Q10 (CoQ10) was reincorporated into CoQ10-depleted membrane preparations from heart mitochondria by two methods reported to achieve full restoration of reduced nicotinamide-adenine dinucleotide (NADH) oxidase activity. Reincorporation in aqueous medium or in an anhydrous one were compared on the basis of restoration of NADH and succinoxidase activities, effect on NADH dehydrogenase activity, CoQ10 content, and reactivity with piericidin A. While reversible removal of CoQ10 from the succinoxidase system is readily achieved without obvious damage to the dehydrogenase in the reconstituted particles, NADH dehydrogenase is significantly modified even though there is extensive restoration of NADH oxidase activity. Contrary to previous evidence, reincorporation of CoQ10 to the level originally present does not result in full restoration of NADH oxidase activity. Comparison of NADH and succinoxidase activities on titration of the depleted particles with increasing amounts of CoQ10 further suggests a compartmentation of CoQ10 at the flavoportein junction, a proposal supported by the findings of Ernster and coworkers from experiments involving gradual depletion of the particles by serial pentane extractions. The observation that in reconstituted particles the inhibition by piericidin A is competitive with respect to CoQ10 is discussed in relation to the mechanism of action of this inhibitor.
Article
1. Durohydroquinone (H2DQ) has been used to provide pulses of reducing equivalents to suspensions of pigeon heart mitochondria and submitochondrial particles.2. H2DQ oxidation in the presence of pentachlorophenol was 95% sensitive to antimycin A and 99% sensitive to cyanide.3. Pulses of 150 μM H2DQ produced reduction of cytochromes b (60–90% of total absorption), fluorescent flavoprotein (25% of total fluorescence) and pyridine nucleotide (60% of total fluorescence). Reduction of the latter two components was mediated through reversed electron transfer and was energy dependent.4. Pulses of 10–30 μM H2DQ brought about redox cycles of ubiquinone, cytochrome(s) b and absorbing flavoprotein (succinate dehydrogenase). Addition of antimycin A altered the kinetics of electron equilibration between ubiquinone and cytochrome(s) b. Antimycin A enhanced the efficiency of reduction of cytochrome(s) b and flavoprotein by 3–20 μM H2DQ, but diminished the efficiency of ubiquinone reduction under the same experimental conditions. In the absence of inhibitor, ubiquinone was quantitatively the most important acceptor for the electrons arising from H2DQ. In the presence of antimycin A, cytochrome(s) b was the most important acceptor for H2DQ.5. In the presence of antimycin A, ubiquinone did not show redox cycle changes synchronous with the redox cycles of flavoprotein and cytochromes b, and thus seem not to be an obligatory member for electron transfer in the main pathway from succinate dehydrogenase-cytochrome(s) b to oxygen. This antimycin A effect is interpreted as the result of a conformational change of cytochrome(s) b or an alteration of the hydrocarbon core of the membrane that ubiquinone occupies.6. 1.2–15 μM additions of H2DQ to cyanide-blocked mitochondria or submitochondrial particles brought about pulses of reducing equivalents which were distributed among the oxidized carriers according to the redox potential of these components. Cytochromes a + a3 and c, absorbing flavorprotein and ubiquinone titrated in the presence or absence of uncoupler, as homogeneous pools in a sequence according to their reported potential values. Cytochrome(s) b titrated in the absence of uncoupler as a heterogeneous pool and in the presence of uncoupler as a homogeneous pool in the latter case the absorbance reaching 50% of the total.7. In the aerobic steady state (plus or minus ATP) a pulse of 250 μM H2DQ reduces cytochromes b absorbing at 562 and 557 nm. The form absorbing at 557 nm is postulated to be cytochrome b555.
Article
The primary event of coupled electron transfer at phosphorylation site II is identified with a modification in one of the two chemically distinct forms of cytochrome b, designated as the energy-transducing cytochrome b(T). This modification is expressed through a change in the redox midpoint potential and by an increase in its reaction half time with cytochrome c(1). In pigeon heart mitochondria cytochrome b(T) exhibits an absorption maximum at 564 nm and on this basis, it can be distinguished from Keilin's cytochrome b which exhibits an absorption maximum at 560 nm and serves as an electron carrier on the substrate side of cytochrome b(T). Kinetic capability of cytochrome b(T) is evidenced by its rapid electron transfer and energization time of less than 200 msec, its thermodynamic capability-by a 280 mV potential span suitable for providing one of the two electron transfer reactions required in ATP formation. Two secondary events of coupled electron flow may be identified with a charge separation across the lipid structure of the permeability barrier and a change in water structure; both events result in an increased 1-anilino-8-naphthalene-sulfonic acid (ANS) response to the altered environment.
Article
The standard redox potential of ubiquinone in submitochondrial particles is measured by equilibration with the succinate/fumarate system to about 65 mV at pH 7, with the redox ratio, n= 2. The pH dependence is -60 mV/Δ pH. The lower value measured for bound ubiquinone is discussed in comparison with the value found for isolated ubiquinone (104 to 112 mV, pH 7) determined in ethanol-HCl. In parallel experiments the standard redox potential of cytochrome b was measured to 72.5mV at pH 7. The pH dependence is ΔE/Δ pH = 0 at pH < 6.8 and - 60 mV ΔHp at pH > 6.8.