genova_2011 new developments on the functions of coenzyme q in mitochondrial - critical review

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Critical Review

New developments on the functions of coenzyme Q in mitochondria

Maria Luisa Genova* and Giorgio Lenaz

Dipartimento di Biochimica ‘‘G. Moruzzi’’, Università di Bologna, Bologna, Italy

Abstract.The notion of a mobile pool of coenzyme Q (CoQ) in the

lipid bilayer has changed with the discovery of respiratory

supramolecular units, in particular the supercomplex

comprising complexes I and III; in this model, the electron

transfer is thought to be mediated by tunneling or

microdiffusion, with a clear kinetic advantage on the transfer

based on random collisions. The CoQ pool, however, has a

fundamental function in establishing a dissociationequilibrium with bound quinone, besides being required for

electron transfer from other dehydrogenases to complex III.

The mechanism of CoQ reduction by complex I is analyzed

regarding recent developments on the crystallographic

structure of the enzyme, also in relation to the capacity of

complex I to generate superoxide. Although the mechanism

of the Q-cycle is well established for complex III,

involvement of CoQ in proton translocation by complex I is

still debated. Some additional roles of CoQ are also

examined, such as the antioxidant effect of its reduced form

and the capacity to bind the permeability transition pore

and the mitochondrial uncoupling proteins. Finally, aworking hypothesis is advanced on the establishment of a

vicious circle of oxidative stress and supercomplex

disorganization in pathological states, as in

neurodegeneration and cancer.

VC 2011 International Union of Biochemistry and Molecular Biology, Inc.Volume 37, Number 5, September/October 2011, Pages 330–354 E-mail: [email protected]

Keywords: coenzyme Q, mitochondria, respiratory chain,supercomplexes

1. Introduction: Physical and

chemical properties of coenzyme QProgress and new developments of research in recent yearshave somewhat modified our understanding of the bioener-getic function of coenzyme Q (CoQ). This review is an updateof the role of CoQ in mitochondria; therefore, other functionsrelated to extra-mitochondrial locations are not consideredhere. To this purpose, it is useful to briefly remind someproperties that are relevant to its function [1].

CoQ exists in three redox states, fully oxidized (ubiqui-none, Q), semiquinone (ubisemiquinone, SQ), and fullyreduced (ubiquinol, QH2 ), but the existence of different pos-sible levels of protonation increases the possible redox

forms of the quinone ring. Because of its extreme hydropho-bicity, natural CoQ (CoQ 10 in humans) can be present inthree physical states: forming micellar aggregates, dissolvedin lipid bilayers, and bound to proteins. Although the formerstate may be experimentally important [2], in the living cellCoQ is distributed among the other two states.

The extent to which CoQ is bound to mitochondrial

proteins is an important parameter in relation to its function.If we consider bound CoQ in a 1:1 stoichiometry with thecomplexes interacting with the quinone (complex I, complex II, and complex III), in beef heart mitochondria we come upto about 0.35 nmol/mg protein and, therefore, we mustassume that most CoQ ( >84%) is free in the bilayer. A directstudy [3] of the amount of CoQ bound to mitochondrialproteins in five different mammalian species showed thatthe protein-bound aliquot ranges between 10 and 32% of total CoQ.

It has been assumed for long time that the shape of the CoQ molecule is linear, with some possibility of rotationallowed for the long isoprenoid tail. Bending of the molecule

[4] is confirmed by linear dichroism studies [5] of its locationin the hydrophobic midplane of the lipid bilayer, with the po-lar head oscillating about the third isoprene unit betweenthe midplane (wholly linear shape) and the polar heads of the phospholipids (maximal bending of 90 ). The modelallows for movement of the redox center of CoQ that isrequired for interaction in the mitochondrial complexes withpartner redox centers, which are situated at the level of thehydrophilic heads of the phospholipids [6,7] (Fig. 1A).

A computer simulation of molecular dynamics of CoQ homologs in the vacuum showed that the lowest energylevel is characterized by a folded conformation, where the

*Address for correspondence: Prof. Maria Luisa Genova, Ph.D., Dipartimento diBiochimica ‘‘G. Moruzzi,’’ Via Irnerio 48, 40126 Bologna, Italy. Tel.: þ39-051-2091214.Fax: þ39-051-2091224; E-mail: [email protected] 4 April 2011; accepted 6 April 2011DOI: 10.1002/biof.168

Published online 11 October 2011 in Wiley Online Library (wileyonlinelibrary.com)

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polar head is in tight contact with the last isoprenoid unit of the hydrophobic tail [8] (Fig. 1B). The folded conformationwas found for both oxidized and reduced quinones, in gen-eral with small energy differences between the oxidized andreduced forms. The folded conformation may also apply toCoQ in the hydrophobic interior of natural membranes, as

supported by the experimental demonstration by magneticresonance techniques that ubisemiquinones are folded in or-ganic solvents [9]. A neutron diffraction study [10] localizedCoQ 10 in the membrane midplane, although the actual shapecould not be defined.

A folded structure of CoQ has important implications.First, the similar size of short and long homologs wouldexplain the similar high rates of lateral diffusion for all qui-none homologs [2]. In addition, protein binding during elec-tron transfer may require unfolding, contributing to the highactivation energy and low collision efficiency observed forelectron transfer ( e.g., ref. 11).

The cyclohexane/water partition coefficients of differ-ent quinones are good parameters of their hydrophobicities[12,13]. An additional consequence of the high hydrophobic-ity of ubiquinones, related to their partition coefficients, istheir extent of solubility in monomeric state [14,15]; waterinsolubility is particularly a serious phenomenon for oxidizedquinones, as in complex I activity determination. An impor-tant consideration concerning the use of ubiquinones assubstrates is their reaction with the partner enzymes fromwithin the lipid bilayer: a method to determine the truemembrane K m and the partition coefficients was describedby steady-state kinetic measurements at varying phospho-lipid fractional values in the assay medium [16].

A datum of particular interest would be the K D of CoQ from the partner respiratory complexes to the lipid bilayer. Anindication may come from the apparent K m obtained for CoQ 10in integrated respiratory activities, e.g., succinate-cytochromec reductase through integration of complex II and complex IIIin extraction and reconstitution studies [17]; however, theseK m in the composite system are poised functions of V max and

dissociation constants for CoQ of the complexes involved;these ‘‘K m’’ can, therefore, be varying with rate changes of thecomplexes linked by the CoQ pool [18,19].

The lateral diffusion of quinones in lipid bilayers hasreceived particular attention in relation to their role in elec-tron transfer processes in the mitochondrial respiratory chain;according to the ‘‘random collision model’’ of electron trans-fer proposed by Hackenbrock et al. [20], all components of the mitochondrial respiratory chain are randomly distributedin the plane of the membrane and undergo independent lat-eral diffusion; the mobility of the smaller components, suchas CoQ and cytochrome c, is faster than that of the macromo-lecular complexes and assures electron transfer by random

collisions with the latter. In addition, they suggested thatCoQ diffusion in the mitochondrial membrane is the rate-lim-iting step in the whole electron transfer process.

A variety of techniques have been used to measure thelateral diffusion in artificial lipid bilayers and in natural mem-branes, yielding a broad range of values for the diffusion coef-ficients D l. Collision-dependent methods measure lateral diffu-sion to distances of several nanometers and include excimerformation or fluorescence quenching, whereas methods basedon the redistribution of probe molecules measure diffusion ona micrometer scale for which the most versatile technique isfluorescence recovery after photobleaching (FRAP). Exploitingthe FRAP technique with fluorescent-labeled ubiquinone ana-

logs, diffusion coefficients were calculated in mitochondrialmembranes in the range of 109 cm2/sec [20,21]. On the otherhand, exploiting collisional fluorescence quenching by nonmo-dified oxidized CoQ homologs of membrane-partitioned fluoro-phores, Fato et al. [2] calculated diffusion coefficients >106

cm2/sec in both liposomes and mitochondrial membranes.The FRAP technique requires chemical modification of the

CoQ molecule by covalent binding of fluorescent reportergroups, thus increasing the size and hydrophilicity of CoQ; thus,the presence of hydrophilic fluorophores bound to the CoQ mol-ecules in the FRAP experiments throws some doubts on theexcessively low values for Dl found using that technique [4].

Fig. 1. Molecular models of coenzyme Q 10. A: Possibledispositions of the long-chain quinone as a moleculeintercalated between the two lipid monolayers, whosepolar head can oscillate about the third isoprene unitbetween the midplane (wholly linear shape) and thehydrophilic heads of the phospholipids (maximalbending at 90 ). B: Folded conformation of ubiquinone-

10 (left) and ubiquinol-10 (right) as predicted bymolecular dynamics simulation. [Color figure can be viewed in the online issue, which is available atwileyonlinelibrary.com.]

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2. Structure of the respiratory chain:Respiratory supercomplexes, a newchallenge for the mechanism andcontrol of oxidative phosphorylation

2.1. The discovery of supercomplexesA retrospective analysis of the literature reveals that the idea

of supramolecular associations between respiratory enzymeswas present since the early times: the original view derivedfrom the spectrophotometric pioneering studies of Chanceand Williams [22] depicted the respiratory chain as a solid-state assembly of flavins and cytochromes in a protein matrix.

Circumstantial evidence against a random distributionof respiratory complexes also came from the early investiga-tions reporting isolation of complex I–complex III [23] andcomplex II–complex III units [24], indicating that such unitsmay be preferentially associated in the native membrane.

Much more recently, new evidence of multicomplex units in yeast and mammalian mitochondria was obtainedintroducing blue native polyacrylamide gel electrophoresis

(BN-PAGE) [25]. In bovine heart mitochondria, complex I–IIIinteractions were apparent from the presence of supercom-plexes also comprising either complex III alone or both com-plexes III and IV at different copy numbers (respirasomes),and only 14–16% of total complex I was found in free formin the presence of digitonin [26], so it seems likely that allcomplex I is bound to complex III in physiological conditions,in the absence of detergents. On the other hand, knowingthe accurate stoichiometry of oxidative phosphorylation com-plexes [26], it is plausible that approximately one-third of total complex III in bovine mitochondria is not bound tomonomeric complex I. The fraction of complex IV in freeform represents >85% of total cytochrome oxidase of mito-chondria. Associations of complex II with other complexes of the OXPHOS system could not be identified, although smallamounts of supercomplexes comprising also complex II wererecently described [27,28]. Moreover, the results of Wanget al. [29] provide evidence of a multifunctional fatty acid b-oxidation (FAO) complex within mitochondria that is physi-cally associated with OXPHOS supercomplexes and promotesmetabolic channeling of electrons directly from FAO to therespiratory chain. To examine the kinetic of the bridgingreaction, in vitro assays were performed using palmitoyl-CoAas substrate in a reaction mixture containing excess cyto-chrome c, CoQ, and a sucrose gradient fraction of mitochon-

drial proteins enriched in respiratory supercomplexes (alongwith KCN to inhibit electron transfer from cytochrome c tocomplex IV); under these conditions, reduction of cyto-chrome c was efficiently promoted, indicating that the neces-sary FAO components were also present in the fraction tomediate oxidation of acyl-CoAs with no accumulation of pathway intermediates.

2.2. Molecular structure of supercomplexesThree-dimensional models of the I1III2 supercomplex isolatedfrom plant [30,31] and mammalian mitochondria [32,33] were

generated by comparison of the 2D projection map of thesupercomplex, as revealed by electron microscopy (EM)analysis and single particle image processing, with knownEM and X-ray structures of complex I and complex III. Posi-tions and orientations of all the individual complexes weredetermined in detail in a bovine supercomplex consisting of complex I, dimeric complex III, and complex IV (I1III2IV1 );Schäfer et al. [32–34] demonstrated that complexes III andIV are both associated with the membrane arm of complex Iand in contact with each other.

It has also been proposed that the OXPHOS complexesmay assemble into higher types of organization, formingrow-like megacomplexes composed by supercomplexes asbuilding blocks, which seem to be important for the mor-phology of the inner mitochondrial membrane [35–37].

Systematic chemical studies aimed to directly deter-mine the contacting subunits and the protein–proteininteraction sites of associated complexes are needed togain substantial progress in the molecular structure of supercomplexes. Early crosslinking investigations of bovine

heart mitochondria showed that it was not possibleto purify complex IV independently of complex III and toseparate cytochrome c from complex IV [38]. In addition,intercomplex crosslinkings were observed for the quinol-cytochrome c reductase and cytochrome c oxidase in qui-nol-oxidase supercomplex isolated from the thermophilicBacillus PS3 [39]. To our knowledge, no study of this kindhas ever been performed dealing with complex I and itsputative partners.

2.3. Kinetic evidence of supercomplex organizationThe biochemical characterization of the supercomplexes is

still poor and mostly obtained by indirect observations of deviations from ‘‘pool’’ behavior of electron transfer and fromstudies aimed to prove substrate channeling by metabolic flux control analysis; on the other hand, very few studies are avail-able on the kinetic properties of supercomplexes.

2.3.1. Deviations from pool behavior. The notion of the CoQ pool as the mechanism for integrated electron transfer fromdehydrogenases to cytochromes, described by the hyper-bolic relationship between the observed rate of electrontransfer of the entire respiratory chain and the rate of eitherreduction or oxidation of CoQ [40,41], has been widely

accepted and has gained place in all biochemistry textbooks;nonetheless, deviations from pool behavior have also beendescribed, raising doubts on the universal validity of the hy-pothesis [18].

The nonhomogeneity of the ubiquinone pool withrespect to succinate and NADH oxidation [42] may be inter-preted today in terms of compartmentalization of CoQ in theI–III supercomplex in contrast with the free pool used forconnecting complexes II and III.

Gutman [42] also investigated the properties of theNADH and succinate oxidation in submitochondrial particlesin relation to the rates of energy-dependent reverse electron

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transfer from succinate to NADþ and of forward electrontransfer from NADH to fumarate, concluding that ‘‘the elec-tron flux from succinate dehydrogenase to oxygen (forwardelectron transfer toward complex III) or to NADH dehydro-genase (reverse electron transfer) uses the same carrier andis controlled by the same reaction,’’ whereas ‘‘the electrontransfer from NADH to oxygen does not share the samepathway through which electrons flow in the NADH-fumaratereductase.’’ In other words, complex I and complex II arelinked by a different pathway with respect to complex I andcomplex III, which is in line with what is found in the super-complex organization.

Kröger and Klingenberg [40,41] already noticed that10–20% of CoQ in submitochondrial particles is not reducedby any substrate. More recently, Benard et al. [43] describedthe existence of three different pools of CoQ in rat liver andmuscle mitochondria: one pool is utilized during steady-state respiration, another is mobilized as a reserve in caseof a perturbation to maintain the energy fluxes at normalvalues ( e.g., due to inhibition of the respiratory complexes

or in case of mitochondrial diseases), and a third one is notmobilizable and is unable to participate in succinate-depend-ent respiration. The reserve of CoQ was about 8% in muscleand 23% in liver, whereas the nonutilizable CoQ was about79% in muscle and 21% in liver. These results are compati-ble with CoQ compartmentalization, although similar resultswith NADH oxidation were not provided.

2.3.2. Flux control analysis of the respiratory complexes.Metabolic flux control analysis [44] assumes that the individ-ual steps of a composite pathway consist of separateenzymes joined by the diffusion of common intermediates.

In any such a system, the control would be differentlyexerted by one or more steps in the pathway, but the sumof the control coefficients of all steps would approach 1.

On the other hand, in enzymatic supercomplexeswhere the interactions between active sites are fixed andsubstrates and intermediates are channeled from one site toanother one without leaving the protein environment, themetabolic pathway would behave as a single enzyme unit,and inhibition of any one of the enzyme components wouldelicit the same flux control, so that the sum of all apparentcoefficients would be above 1 [45].

In particular, in a system in which the respiratory chainis totally dissociated from other components of the OXPHOS

apparatus ( i.e., ATP synthase, membrane potential, and car-riers), such as open nonphosphorylating submitochondrialparticles, the existence of a supercomplex would elicit a flux control coefficient near unity at any one of the respiratorycomplexes.

Performing titrations of enzyme inhibition using spe-cific inhibitors of each complex [46], both complexes I andIII were found to be highly rate controlling over NADH oxida-tion, a strong kinetic evidence suggesting the existence of functionally relevant association between the two complexes.On the contrary, complex IV appeared to be randomlydistributed. Moreover, complex II was fully rate limiting for

succinate oxidation, clearly indicating the absence of sub-strate channeling toward complexes III and IV.

In permeabilized mitochondria from freshly harvestedpotato tubers, inhibitor titration experiments indicate thatcomplexes III and IV are involved in the formation of asupercomplex assembly comprising complex I, whereas thealternative dehydrogenases, as well as the molecules of complex II, are considered to be independent structureswithin the inner mitochondrial membrane [47].

Flux control analysis in intact mitochondria under phos-phorylating or uncoupled conditions usually exhibits low flux control coefficients for respiratory complexes in mitochon-dria isolated from various tissues ( cf. ref. 1) because thecontrol is distributed among other components, as the ade-nine nucleotide carrier, the ATP synthase, and presumablythe substrate carriers and the NAD-linked dehydrogenases.Nevertheless, we have obtained evidence of the existence of a complex I–III association in mitochondria under phospho-rylating conditions by exploiting flux control analysis infreshly prepared liver mitochondria from old rats [47] where,

being respiration rate limiting over other accessory activitiesbecause of aging, we were able to detect high control byboth complex I and complex III.

Figure 2 shows in a schematic way the difference exist-ing between the collision-based model and the supercom-plex organization of the respiratory chain.

2.3.3. Electron transfer activity of isolated supercomplexes.Supercomplexes separated by BN-PAGE and related techni-ques are active for what concerns their component individ-ual complexes, as shown by in-gel catalytic activity assays[32,49]. As supercomplexes can be isolated after mild solu-

bilization [50,51], they become amenable to investigation of their integrated activity. Stroh et al. [50] first demonstratedthat the respirasome isolated from P. denitrificans hasNADH cytochrome c reductase and NADH oxidase activities,showing that the supercomplexes are active and presum-ably exert channeling of the ‘‘mobile components’’ CoQ andcytochrome c; indeed, CoQ was found enriched 10-foldin the supercomplex, whereas the low NADH oxidaseactivity suggested some loss of cytochrome c from therespirasome.

Acı́n-Pérez et al. [27] confirmed the presence of differ-ent forms of supercomplexes after solubilization of mouseliver mitochondria in different detergents and BN-PAGE; at

difference with previous studies, some supercomplexes alsocontained complex II and ATP synthase (complex V). Oneparticular subfraction (band 3) contained all complexes I, II,III, and IV and in addition also cytochrome c and CoQ 9. Theband was excised from the gel and showed full respiratoryactivity from either NADH or succinate, which was sensitiveto the specific respiratory inhibitors of all involved com-plexes. Therefore, the oxygen consumption shown by thesupercomplex bands is not only the consequence of thepresence of all the respiratory complexes and ‘‘mobile’’ car-riers needed but also reflects the proper arrangement into afunctional structure.



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Fig. 2. Schematic models showing ubiquinone as a mediator of the electron transport in the respiratory chain. A: Schemeof flux control in two models of NADH-dependent respiration by the mitochondrial respiratory chain. The flux-summationtheorem, a central principle of metabolic control analysis, describes how the control of flux through any metabolicpathway of arbitrary complexity is distributed among the component reaction steps. (Left) In a linear pathway composed of a series of enzymes connected by mobile intermediates, i.e., CoQ and cytochrome c, the relative control of each respiratorycomplex may be different and the sum of all the flux coefficients ( C i ) is equal to unity. (Right) In a supercomplex, any stepin the obligatory path is regarded as a component of a single enzyme and must be completely rate controlling; therefore,

each component elicits maximal flux control coefficient (C i 5 1) and the sum of all coefficients results in a value higher than 1. B: Effect of antimycin A (AA) inhibition on the overall velocity of respiration according to pool behavior or tochanneling in a supercomplex organization. Left, pool behavior: coenzyme Q is shown as a freely diffusible interconnecting mediator (Q pool) among the electron donors (complex I) and the acceptors (complex III). Interaction of AA with therespiratory chain causes the ‘‘nonlinear’’ inhibition curve of substrate oxidation (due to multiple choices for quinoloxidation by the residual active molecules of complex III, as indicated also by the dotted arrows). As shown by Krö ger andKlingenberg [41], the relation between the respiratory activity (V obs/V 0 ) and the amount of antimycin relative to theantimycin titer (i 0/n0 ) depends on the ratio V red/V ox ; the most hyperbolic curve is observed with the smallest value (i.e.,V red/V ox 0.05). Right, supercomplex arrangement: the serial mediation by ubiquinone (Q) only within the I–IIIsupramolecular assembly would result in a linear relation of the respiratory activity to antimycin. V 0 5 V obs in the absenceof antimycin; V ox , rate of ubiquinol oxidation; V red, rate of coenzyme Q reduction; V obs, overall flux observed. Picture wastaken from ref. 48. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

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This finding directly confirms that supercomplex orga-nization is compatible with electron transfer and, however,does not discard the idea that electron transfer is possiblein the membrane in the absence of supercomplex organiza-tion in accordance with the random collision model. In fact,Acı́n-Pérez et al. confirmed that most or all complex I isindeed in the form of supercomplex, but most of complexesII, III, and IV appear to be free as isolated complexes. Theauthors proposed a ‘‘plasticity model’’ where both types of organization are possible and functional and depend on thedifferent mitochondrial systems and on the particular physi-ological states. The plasticity model well suits the informa-tion obtained by us by flux control analysis, suggesting thatelectron transfer between complex I and complex III iseffected only by CoQ channeling whereas that between com-plexes II and III and complexes III and IV seems to occurmostly by the pools of CoQ and cytochrome c, respectively(at least in beef heart and rat liver mitochondria). We willfurther discuss this issue in Section 9 à propos of pathologi-cal changes.

3. Is there a function for thecoenzyme Q pool?

There is no doubt that a mobile pool of CoQ exists in theinner mitochondrial membrane and that this pool coexistswith protein-bound CoQ. Is this pool just a reservoir of anexcess of CoQ molecules without a specific function or is thepool necessary for functioning of the respiratory chain and/or for additional functions?

3.1. Dissociation equilibrium of bound componentsAs previously described, complex I is almost totally associ-ated in a supercomplex with complex III, with electronchanneling of bound CoQ in the boundary between thetwo complexes. However, this does not exclude that freeCoQ in the pool is also necessary for proper channelingbetween the complexes. In fact, the bound intercomplex quinone that allows electron flow directly from complex Ito complex III must be in dissociation equilibrium with theCoQ pool, so that its amount, at steady state, would bedictated by the size of the pool; this equilibrium explainsthe saturation kinetics for total ubiquinone exhibited by

the integrated activity of complex I and complex III [17]and the decrease of respiratory activities in mitochondriafused with phospholipids with subsequent dilution of theCoQ pool [52]. To be in agreement with the experimentalobservation obtained by metabolic flux analysis, this prop-osition requires that the dissociation rate constants ( k off )of bound CoQ be considerably slower than the rates of intercomplex electron transfer via the same bound quinonemolecules.

By this way, free CoQ acts as a reservoir for binding tothe I–III supercomplex; in addition, free CoQ may be a reser-voir for other functions believed to require CoQ binding to

specific proteins, such as uncoupling proteins (UCPs) [53]and the permeability transition pore (PTP) [54] (see Sections7 and 8).

A different question is whether electron transferbetween complex I and complex III can occur via the CoQ pool in the absence of supercomplex organization. Analysisof the literature does not offer clearcut examples of electronflow between complexes I and III in mitochondrial mem-branes mediated with certainty by the CoQ pool. Studies of respiration in pathological conditions [55,56] showed thatelectron transfer in the absence of supercomplex organiza-tion is lost even if activity of the individual complexes is nor-mal. Early reconstitution studies, however, had indicatedthat electron transfer is possible in both modes; the associa-tion of complex I with complex III [57,58] allows both chan-neling (electron transfer stoichiometric with the percentageamount of complex III associated to complex I) and CoQ pool behavior (hyperbolic relation). In reconstitution studiesof complexes I and III in phospholipid vesicles [47], NADH-cytochrome c reductase activity follows pool behavior at pro-

tein dilutions with phospholipids higher than 1:10, whereasat lower dilution pool behavior is not effective any more.The discrepancy between these observations requires furtheranalysis; it is of interest that, in the absence of supercom-plexes, pool behavior has been shown in reconstitutedsystems, whereas lack of electron transfer was observed innative mitochondria, thus a possibility deserving investiga-tion is in a possible constraint existing in the natural mem-brane. It has to be reminded, however, that the papersquoted [55,56] have not investigated the CoQ concentrationin the membrane.

3.2. Electron transfer between individualcomplexes not involved in supercomplex organizationThe CoQ pool is required for electron transfer from complex II to complex III; indeed, complex II kinetically follows poolbehavior after extraction and reconstitution [40,41] and inintact mitochondria [59] in accordance with the lack of super-complexes found by both BN-PAGE and flux control analysis(see previous section). The existence of small amounts of supercomplexes containing complex II, described by Acı́n-Pérez et al. [27], does not contradict the knowledge thatmost of succinate oxidation takes place by the CoQ poolbetween complexes II and III. An exception is the study of

Boumans et al. [60] in yeast mitochondria, where succinateoxidation resumed pool behavior only in the presence of chaotropic agents.

Furthermore, other enzymes, such as glycerol-3-phos-phate dehydrogenase, ETF dehydrogenase, dihydroorotatedehydrogenase, and choline dehydrogenase, which are likelyto be in minor amounts and strongly rate limiting in inte-grated electron transfer, are probably inserted in the respira-tory chain by interaction through the CoQ pool [1]. A studyaddressed to this problem [61] demonstrated that in brownfat mitochondria the inhibition curve of glycerol phosphate-cytochrome c reductase is sigmoidal in the presence of



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3.3. Saturation kinetics: Is CoQ mitochondrialconcentration saturating for electron transfer?In previous studies, we have discussed the relation existingbetween rate of respiration and CoQ membrane concentra-tion Qt [19]; the rate is hyperbolically related to [Qt], andmaximal turnovers of electron transfer are attained only at[Qt] saturating both rates of reduction and oxidation of the

quinone pool. The ‘‘K m’’ derived by the complex equationdescribing this composite system [18,19] is a poised functionof V max and dissociation constants for CoQ of the complexesinvolved; this ‘‘K m’’ can, therefore, be varying with ratechanges of the complexes linked by the CoQ pool, but isanyway an important parameter, in that it is operationallydescribed as the Qt concentration yielding half-maximalvelocity of integrated electron transfer.

The relation between electron transfer rate and CoQ

concentration was seen for NADH and succinate oxidation in

reconstituted systems and in phospholipid-enriched mito-

chondria [17,52,64]. Direct titrations of CoQ-depleted mito-

chondria reconstituted with different CoQ supplements

yielded a ‘‘K m’’ of NADH oxidation for Qt in the range of 2–5nmol/mg mitochondrial protein [17], corresponding to a Qtconcentration of 4–10 mM in the lipid bilayer. A puzzling

observation is that the K m for CoQ 10 of NADH-cytochrome c

reductase is much higher than that of succinate-cytochrome

c reductase. Analysis of the literature shows that the physio-

logical CoQ content of several types of mitochondria [65] is

in the range of the K m for NADH oxidation and, therefore,

not saturating for this activity.It has been demonstrated that incubation of beef heart

submitochondrial particles in a CoQ 10 solution leads to incor-poration of CoQ 10 in their membranes [66]. The same

authors found that kinetic saturation with CoQ 10 could notbe achieved because of the intrinsic insolubility of the mole-cule, thus concluding that the upper limit of electron trans-fer from NADH is a function of CoQ 10 solubility in the mem-brane phospholipids. Nevertheless, a soluble formulation of CoQ 10 (QterVR ) is able to enter mitochondria in cultured cellsenhancing respiratory activity [67].

Attempts to increase CoQ concentration and respiratoryactivity in mitochondria by exogenous supplementation of the quinone in humans have been successful in cases of mitochondrial genetic diseases characterized by CoQ defi-ciency, but the studies have shown substantial difficultiesdue to pharmacokinetic constraints preventing efficient

uptake by cells and mitochondria with requirement of extremely high dosage [68]. Rosenfeldt et al. [69] usedCoQ 10 in cardiac surgery patients receiving 300 mg/dayorally of CoQ 10 dispensed in soy bean oil demonstrating afourfold increase in serum concentration of CoQ 10 and a 2.5-fold increase in of CoQ 10 in atrial myocardium, whichincluded a 2.4-fold increase of CoQ 10 in atrial mitochondria.The coupled respiration was lower in the mitochondria fromCoQ 10-treated patients; however, the ADP:O ratios were sig-nificantly higher. It should be borne in mind that there aremultiple rate-limiting steps in coupled respiration [70], sothat the extent of CoQ 10 deficiency and its correction by

exogenous quinone may not be apparent if a rate-limitingstep involves a reaction where CoQ is not involved.

4. Mechanism of coenzyme Qchanneling in supercomplex I–III

The functional consequence of supercomplex assemblies in

the respiratory chain is substrate channeling in intercomplex electron transfer. Substrate channeling is the direct transferof an intermediate between the active sites of two enzymescatalyzing consecutive reactions [71]; in the case of electrontransfer, this means direct transfer of electrons between twoconsecutive enzymes by successive reduction and reoxida-tion of the intermediate without its diffusion in the bulk me-dium. In such a case, intercomplex electron transferbecomes indistinguishable from intracomplex electron trans-fer, so that the so-called mobile intermediates, predicted toexhibit substrate-like behavior in the classic view of the ran-dom collision model [20], would rather be buried in the

interface between the two consecutive complexes.Kinetic analysis allows distinguishing the channelingfrom the random diffusional encounters: the problem forelectron transfer was tackled for the first time by the pio-neering work of Kröger and Klingenberg [40,41]; subse-quently flux control analysis was exploited by us with theprecise aim of demonstrating channeling.

4.1. Mechanism of channeling: Electron tunnelingor microdiffusion?The fundamental design of electron transfer proteins is twocatalytic sites connected by redox chains [72]; the two catalytic

sites and the connecting chain may be entirely within a singleprotein or belong to different protein subunits. In the respira-tory chain of mitochondria, the redox complexes are composedby several subunits containing a trail of cofactors needed toallow sufficiently short distance for electron transfer to occur.Intraprotein electron transfer is typically limited by tunnelingthrough the insulating protein medium between the edges of the interacting centers: electron tunneling in protein is reason-ably well described by a simple exponential decay with dis-tance, so that the maximal distances allowing physiologicalelectron transfer should not exceed 13–14 Å [73].

Interprotein electron tunneling obeys to the same ex-ponential rate dependence on distance as intraprotein elec-

tron transfer; however, small-scale constrained diffusivemotions are sometimes necessary to bring redox centerswithin the 14-Å tunneling limit; electron transfer rates reflectdiffusional motion of domains of the proteins after a pro-tein–protein complex has been formed [74]. In the case of supercomplexes formed by apposition of individual com-plexes connected by potentially mobile cofactors, what isthe mechanism of electron transfer? Ideally, we should havea detailed knowledge of the molecular structure of the inter-acting sites, and this knowledge is still lacking. Obviously,we may have the extremes from close docking of the activesites with real interprotein tunneling, up to relatively long



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distances that may be covered either by important conforma-tion changes or by restricted diffusion (microdiffusion) of the mobile components within the space between the twoactive sites; all of these alternatives have in common obli-gate channeling between two fixed sites, so that even thelast situation, microdiffusion, would be quite distinguishablefrom pool behavior where the interaction of the mobile com-ponent may stochastically occur with a great number of pos-sible sites reached by random diffusion. Thus, the previouslydescribed kinetic analysis cannot distinguish among differentpossible mechanisms of channeling. In the interactionbetween complex I and complex III within a supercomplex, if the sites are connected by CoQ microdiffusion, it is possiblethat it takes place within a lipid milieu, although we cannotexclude that the sites are put together by movement of CoQ on the protein or by movement of the protein itself. If lipidis involved, then indirect indications may be obtained fromstudies on the temperature dependence of mitochondrialmembrane-bound enzymes ( cf. ref. 1).

5. Coenzyme Q in intracomplexelectron transfer

5.1. Nature of the CoQ-binding sites inmitochondrial complexesThe high-resolution crystal structures of a number of qui-none-reactive membrane proteins have revealed their qui-none-binding sites (Q-sites) and facilitated their analysis[75–77]. These structures and contributions from biochemi-cal, mutagenic, inhibitor binding, EPR, and IR studies [78–80] have highlighted significant variation and no clear rela-

tionship between architectures and mechanisms of quinonebinding, a part from that in the homologous family of ‘‘typeII’’ photosynthetic reaction centers [81]. It is also worth not-ing that significant variability is seen for quinone binding inthe same protein from different species and that different re-dox states of quinones may adopt different conformations inthe binding pockets. As reported in a previous review by theauthors [82], no common architecture or universal catalyticmechanism can be applied to Q-sites in general, and severaldistinct types of Q-sites associated with respiratory electrontransfer complexes can be specified although they can ex-hibit some very general analogous features. In complex II,several amino acid residues, identified by biochemical analy-

sis as critical to the function of the enzyme, have a clearrole in the mechanism of quinone binding and reduction, butcomparison with other respiratory complexes indicates thatthe structural and inhibition patterns observed for theQ-binding site(s) in complex II are different from those incomplexes I and III [76,83].

It is worth mentioning that the number of binding sitesfor ubiquinone in NADH-Q oxidoreductase is an unsolvedquestion subject to intense controversy. Most suggestionswere derived from studies involving labeled inhibitor analogs[84,85] and up to three sites have been proposed in the lit-erature [86,87].

A better understanding has been derived from therecent crystallization of the entire complex I from bacterialand mitochondrial sources.

Efremov et al. [88] have determined the structure of the hydrophobic domain of complex I from E. coli and theentire complex I from Thermus thermophilus. The high-potential cluster N2 is near the cavity between subunitsNqo4 (corresponding to the 49-KDa subunit in mitochondria)and Nqo6 (PSST subunit in mitochondria), which forms partof a larger cavity at the interface of these subunits withhydrophobic subunits Nqo8 (ND1) and the Nqo7, 10, 11 bun-dle (ND3, ND6, and ND4L). The N-terminal b-sheet of Nqo4(residues 26–60) forms part of this cavity, consistent withlabeling of the corresponding region of bovine 49-kDa subu-nit with azidoquinazoline [89]. Subunit Nqo8 was also la-beled with quinone-like inhibitors [90] and is thought toform a part of quinone-binding pocket along with subunitsNqo4 and Nqo6. Thus, cumulative evidence indicates thatthis cavity at the interface of hydrophilic and hydrophobicsubunits provides a quinone-binding site. It is a large cavity,

up to about 30 A˚

across, consistent with it providing a largecommon binding domain, with partially overlapping sites, forvarious quinone-like inhibitors. Cluster N2, the electron do-nor to CoQ, is about 20–25 Å from the expected surface of the lipid bilayer; thus, some movement of the quinone outof the membrane is likely to be required to approach N2,and its extent will depend on the exact location of thebound quinone in the membrane domain. There are severalpossible sites for accommodating the isoprenoid tail, formedeither by subunit Nqo8 alone or at its interface with theNqo7, 10, 11 bundle. In the latter case, the quinone will needto move about 20 Å to approach cluster N2, whereas in theformer the movement does not need to exceed about 10 Å,

which means that the tail of the quinone can still residemostly within the membrane, as expected for a highly hydro-phobic moiety.

In the crystallized mitochondrial complex I from Yarro-wia lipolytica [91], critical residues for interaction with ubiq-uinone and inhibitors are found at the interface of the highlyconserved 49-kDa and PSST subunits. The last iron–sulfurcluster in the chain, N2, is the immediate electron donor forubiquinone. A funnel-like cavity leading from the N-terminalb sheet of the 49-kDa subunit toward tyrosine-144, (tyro-sine-87 in Thermus thermophilus ), which is in the immediatevicinity of cluster N2, has been mapped by site-directed mu-tagenesis [92,93]. It has been shown by detailed structure/

function analysis that ubiquinone binds directly to this fullyconserved tyrosine [94]. Measured perpendicular to themembrane plane, cluster N2 resides 30 Å above the sur-face of the transmembrane core. The distance from theopening of the ubiquinone-binding pocket at the surface of the membrane arm to the electron donor site is 35 Å. Con-sidering that the binding site for ubiquinone must be at adistance allowing electron transfer of cluster N2, the sub-strate must diffuse 25 Å out from the hydrophobic core of the membrane to position the functional head group in theactive site. This distance accounts for much of the extremelyhydrophobic 40-Å-long side chain of nine isoprenoid units in

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ubiquinone that may slide over a hydrophobic ramp as previ-ously proposed [95,96] ( cf. Fig. 4).

5.2. Mechanism of CoQ reduction in complex IThe role of CoQ in the mechanism of electron transfer of respiratory complexes has been the subject of extensiveresearch. The mechanism of Mitchell’s Q-cycle in complex IIIwith a bifurcation of electrons from ubiquinol into two pathsat high and low redox potential, respectively, is now wellconsolidated, and we refer to excellent reviews on the topic[97–104]. On the other hand, the mechanism by which elec-trons are fed from complex I to CoQ is still object of debate;

here, we give a brief account of the state-of-the art.The mechanism of CoQ reduction by complex I is par-

ticularly intriguing, because more than one bound quinonespecies has been assigned to the enzyme; three ubisemiqui-none signals are detectable in the enzyme [105,106].

It is, however, unlikely that a strongly bound nonex-changeable quinone, comparable to QA in the photosyntheticreaction center, is required for complex I activity, becauseCoQ is not present in fully active preparations of the enzyme[107]. Cluster N2 interacts paramagnetically with two semi-quinone species: the fast-relaxing species QNf is onlyobserved in the presence of a membrane potential, whereas

the slow-relaxing species QNs is also seen during uncoupledturnover [108]; it is not known whether the two signalsreflect two semiquinones in different binding sites or twostates of the same molecule [109]. The crystallographicstructure of complex I, however, seems to exclude, althoughdoes not completely rule out, the presence of separate qui-none molecules [94].

The short-range interaction with N2, which is situatedin the peripheral arm, suggests that the quinone head groupis not deeply buried in the membrane, despite the extremehydrophobicity of the long isoprenoid tail. The structure of the side chain of quinones used for reconstitution is specificfor integrated NADH oxidation activity [4] although shortCoQ homologs and analogs can be used as direct electronacceptors from the enzyme [110]. The observation that theK m of complex I for the short homolog CoQ 1 in bovine heartsubmitochondrial particles is reversibly increased withoutany change in V max after depletion of endogenous CoQ 10 bypentane extraction [11] suggests that CoQ 1 may interact withthe enzyme active site either directly or via bound CoQ 10

entered into the site from the pool (or shared from the com-plex I–complex III supercomplex, see Section 2).The actual mechanism of CoQ reduction is not known,

and models have been based mostly on necessity to accommo-date a mechanism for proton translocation [87,111–114] but notbased on actual experimental evidence; Sherwood and Hirst[115] observed no evidence for a reductant-induced oxidation of ubiquinol, therefore excluding a Q-cycle type of mechanism.

It is generally assumed that two electrons are deliv-ered to CoQ by the same iron–sulfur cluster by two consecu-tive electron transfer reactions [111,112].

The recent findings in our laboratory pinpointing twodifferent classes of inhibitors on the basis of their opposite

effects on oxygen reduction to superoxide during forwardelectron transfer in bovine heart submitochondrial particles[116,117] allow to dissect the two separate steps of reduc-tion of Q to QH2, in a bifurcated scheme where the firstelectron is delivered to CoQ with the formation of semiqui-none in a rotenone-sensitive way; semiquinone is thenreduced to quinol by N2 in a stigmatellin-sensitive way. N2is also the donor to oxygen; rotenone does not preventdelivery of one electron to N2 and then to oxygen, whereasstigmatellin prevents electron delivery to either ubisemiqui-none or oxygen.

A further confirmation of this mechanism derives fromthe effect of inhibitors on reduction of the acceptor dichloro-

phenol indophenol (DCIP). Some DCIP is reduced at the levelof FMN, because there is a component insensitive to DPI;another component is sensitive to DPI and must be reducedat the level of CoQ. In fact, both hydrophilic and hydropho-bic quinones enhance DPI-sensitive DCIP reduction. This lasteffect is still allowed in the presence of stigmatellin but notin the presence of rotenone [117].

These findings demonstrate that DCIP is reduced at asite situated between the rotenone and the stigmatellin inhi-bition sites, a further indication for a split pathway of elec-trons at the CoQ-binding site. According to the scheme pre-sented in Fig. 5, DCIP would be reduced by ubisemiquinone,

Fig. 4. Schematic view of the coenzyme Q-binding cavity in complex I. The cavity is located next toiron–sulfur cluster N2 (in black) at the interface of the49-kDa (in blue-gray) and the PSST subunits (incyan-green). The color gradient indicates the position of amino acid residues that are critical (red), less critical(yellow and green), and not critical (blue) for complex Iactivity. The position of tyrosine-144 and histidine-226is indicated. The arrow indicates a possible access pathfor the quinone substrate (Q). The location of theHRGXE motif is highlighted. Reprinted with permissionfrom Tocilescu et al., Biochim. Biophys. Acta, 2010, 1797,625–632, VC Elsevier. [Color figure can be viewed in theonline issue, which is available atwileyonlinelibrary.com.]



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because its formation is rotenone sensitive but stigmatellininsensitive.

The possible existence of two sites involved in CoQ reduction has to be reconciled with the linear pathway of elec-trons along the series of iron–sulfur clusters as first demon-strated by the crystallographic study of Hinchliffe and Sazanov[118] and confirmed by further crystallographic [88,91] and EPR[119,120] studies; our interpretation is not in contrast with theexistence of a linear pathway, because the two electrons deliv-ered to CoQ for its complete reduction could well be providedalternatively by the same cluster if a suitable conformationalchange occurs after the first electron delivery to provide a gat-ing mechanism for the second electron.

5.3. Coenzyme Q and proton translocationThe involvement of CoQ in proton translocation has beenpioneered by Mitchell in its Q-cycle hypothesis [97]. Involve-

ment of CoQ in proton translocation by complex I has beenpostulated, and several models, either cyclic or linear, wereproposed, although only the recent crystallization of thecomplex has offered hints to elucidate the mechanism of proton translocation.

5.3.1. Complex III. According to the most recent versions of

the Q-cycle [121,122], ubiquinol delivers the first electron atthe outer positive site (called site o or P) of the inner mem-brane to the Rieske iron–sulfur protein and hence to cyto-chromes c1 and c; the result is release of two protons in theintermembrane space and formation of an unstable semiqui-none anion (Q ) at the Qo site, which is immediately oxi-dized to ubiquinone by the low-potential cytochrome b566(bL ). The electron is then delivered to the high-potentialcytochrome b562 (bH ) at the internal negative site (site i orN) and then bH is reoxidized by Q at the Qi site, forminganother semiquinone. The cycle is completed by oxidation of a second molecule of QH2. In this second cycle, the electronreleased by bH reduces the semiquinone at the Qi site

regenerating ubiquinol by taking up two protons from thematrix. Thus, the final stoichiometry of the Q-cycle would be

2QH2 þ 2 cyt: c Fe3þ þ 2H þðinÞ ! Q þ QH2

þ 2 cyt. c Fe2þ þ 4H þðoutÞ

that is

QH2 þ 2 cyt: c Fe3þ þ 2HþðinÞ ! Q þ 2 cyt: c Fe

2þ þ 4HþðoutÞ:

5.3.2. Complex I. N2 is the direct electron donor to bound

ubiquinone [106], and this step is linked to proton transloca-tion [123], although the mechanism has been stronglydebated [87,109,111,114,115,124–126]. Most models impliedat least partly CoQ redox chemistry in the mechanism of Hþ

transport. Nevertheless, because all redox groups in theenzyme appear to be located in the hydrophilic arm or atleast at the interface with the hydrophobic arm, direct cou-pling mechanisms appear unlikely to be solely responsiblefor Hþ movements; this implies that the driving force forproton translocation must be transduced over a considerabledistance to the actual pumping process in the membranearm via conformational coupling [124,127].

The recent X-ray structure of complex I allows a better

understanding of the mechanism of Hþ translocation [88].The bacterial L-shaped assembly consists of the alpha-heli-cal model for the membrane domain, with 63 transmem-brane helices, and the known structure of the hydrophilicdomain. The architecture of the complex provides strongclues about the coupling mechanism; the conformationalchanges at the interface of the two main domains may drivethe long amphipathic alpha helix of NuoL (corresponding toND5 in mitochondria) in a piston-like motion, tilting nearbydiscontinuous helices, resulting in proton translocation.

The indirect conformational mechanism driven by thepiston-like movement of the amphipathic helix of NuoL

Fig. 5. Proposed two-step mechanism for electrontransfer from NADH to quinone in complex I and theeffect of rotenone and stigmatellin. The role of hydrophilic (Q 1 ) and hydrophobic (Q) quinones ishighlighted. CoQ 1 can react with the physiologicalubiquinone reducing site and, because of its higher water solubility, it can also react with the electronescape site, increasing superoxide production.Superoxide is mainly generated at N2 that remainsreduced in the presence of rotenone; stigmatellinallows reduction of Q to semiquinone (Q ) preventing further reduction of Q by N2 that remains oxidized,and Q may be reoxidized by DCIP. The possible site of superoxide generation at FMN (fully reduced) is shownwith a thick arrow.

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would open three Hþ channels in the transmembrane helicesof NuoL, NuoM, and NuoN, respectively. The fourth protonwould be translocated by direct coupling through the redox action of CoQ.

Ohnishi and Salerno reported that two distinct EPR-de-tectable semiquinone species also play important roles inNADH-ubiquinone oxidoreductase (complex I). They werecalled SQ Nf (fast-relaxing semiquinone) and SQ Ns (slow-relax-ing semiquinone). It was proposed that Q Nf serves as a‘‘direct’’ proton carrier in the semiquinone-gated protonpump [111], whereas Q Ns works as a converter between one-electron and two-electron transport processes. Recently,Ohnishi et al. [128] presented a revised hypothesis in whichQ Nf plays a role in a ‘‘direct’’ redox-driven proton pump,whereas Q Ns triggers an ‘‘indirect’’ conformation-driven pro-ton pump. Q Nf and Q Ns together serve as (1e

/2e ) con-verter for the transfer of reducing equivalent to the Q-pool.According to this new model, 2Hþ would be transported bythe direct mechanism and 2Hþ by the conformational mecha-nism. Note, however, that the controversy about the exis-

tence of more than one quinone molecule in the complex isstill open (see Section 5.1).

6. Coenzyme Q and generation of superoxide

6.1. Complex IIICape et al. [129] examined the dependence of oxidation rateon substrate redox potential in the yeast cytochrome bc1complex and found that the rate limitation occurs at thelevel of direct one-electron oxidation of quinol to semiqui-none by the Rieske protein. Oxidation of semiquinone and

reduction of cytochrome b or O2 are subsequent, distinctsteps. These experimental results are incompatible with con-formational gating models that produce superoxide by differ-ent rate-limiting reactions from the normal Q-cycle. Amongthe Rieske FeS center, cytochrome bL, and the redox statesof CoQ p, the authors conclude that the direct reductant of molecular oxygen is SQ.

6.2. Complex IThe identification of the oxygen-reducing site in complex Ihas been the subject of extensive investigation, and sev-eral prosthetic groups in the enzyme have been suggested

to be the direct reductants of oxygen. These include FMN,ubisemiquinone, and iron–sulfur cluster N2 ( cf . refs.116,117, and 130).

The findings from our laboratory of two classes of inhibitors with respect to superoxide generation [117] ( cf .Section 5) are difficult to be reconciled with a mechanismwhereby flavin is the electron donor to oxygen; the twoclasses of inhibitors both act downstream of the iron–sulfurclusters in the enzyme but have opposite effects, in that ro-tenone enhances superoxide production, whereas stigmatel-lin inhibits it [117]. Our conclusion is, however, in contrastwith the findings in isolated complex I [131–133] where FMN

is considered the major electron donor to oxygen to formsuperoxide anion. In particular, accurate redox titrations of the electron donor and an EPR study of the different redox centers [131] appear to exclude either FMN semiquinone orany FeS cluster as the source of superoxide, suggesting thatthe fully reduced flavin delivers one electron to oxygen andthe other one to the chain of iron–sulfur clusters. Galkin andBrandt [132] showed that ROS production was still presentin complex I from a mutant of Yarrowia lipolytica lackingiron–sulfur cluster N2, concluding that FMN is directlyinvolved in this activity.

A possible explanation of this discrepancy is thattwo sites for oxygen reduction exist in the complex, repre-sented by flavin and an iron–sulfur cluster; the latter sitewould be predominant in membrane particles, whereasthe former one might be made better available after com-plex I isolation. The role of supercomplex organization inshielding/opening different sites in the enzymes cannotbe overlooked [1].

Ohnishi et al. [134] presented a new hypothesis that

the generation of superoxide reflects a dynamic balancebetween the flavosemiquinone (semiflavin or SF) and thesemiquinone (SQ). In a purified preparation of complex I,during catalytic electron transfer from NADH to DBQ, thesuperoxide generation site was mostly shifted to the SQ.A quinone-pocket binding inhibitor (rotenone or piericidinA) inhibits the catalytic formation of the SQ, and it enhan-ces the formation of SF and increases the overall superox-ide generation. This suggests that if electron transfer wasinhibited under pathological conditions, superoxide gener-ation from the SF would be increased. The identification of SQ rather than N2 as the electron donor to oxygen is,however, in contrast with the findings reported in the pre-

vious section showing that when SQ reduction to QH2 isblocked by stigmatellin no superoxide is produced [117].Moreover, studies in CoQ-depleted and reconstituted mito-chondria indicated that endogenous CoQ is not requiredfor superoxide generation [130]. It is worth noting thatreconstituted mitochondria, containing a large excess of CoQ 10, produce the same amount of superoxide as CoQ-depleted mitochondria, indicating that endogenous CoQ 10is not a source of ROS.

Although endogenous SQ does not appear to be areductant of oxygen to produce superoxide in complex I,added short-chain quinones have been shown to enhancesuperoxide generation. Early experiments proved the involve-

ment of complex I in ROS production [135]; addition of eitherNADH at low concentration or of NADPH, which feeds theelectrons at decreased rate into the complex, led to copiousROS production detected by lipid peroxidation; addition of NADH at high concentration, but in the presence of rote-none, also induced peroxidation. Water-soluble CoQ homo-logs used as electron acceptors from isolated complex Istimulated H2O2 production, whereas CoQ 6 and CoQ 10 wereinactive [136]. The prooxidant action of hydrophilic quinoneshas been confirmed [117,130,137]; they are thought to bereduced by a one-electron mechanism either by reducedflavin [137] or by N2 [117,130].



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7. Coenzyme Q and UCPs

In the last decade, an exciting topic of research about CoQ has been its role as a cofactor in proton transport by UCPs,the mitochondrial carriers that short-circuit the proton-motive force generated by the respiratory chain and conducethe protons across the inner membrane. The presence of UCPs has been demonstrated also in some nonphotosynthe-

sizing unicellular eukaryotes, including amoeboid and para-site protists, as well as in nonfermentative yeast and fila-mentous fungi; in all cases, the activity of UCPs leads to thesame final effect, i.e., competition for a proton electrochemi-cal gradient with ATP synthase and, thus, diversion of energy from oxidative phosphorylation. Fermentative yeast(such as Saccharomyces cerevisiae ) seem to be the onlyexception in eukaryotes not possessing UCP; in that case,there are no orthologs of the known members of the UCPfamily and no evidence for the presence of UCP-like activitystimulated by free fatty acids (FFAs) and inhibited by purinenucleotides (PNs) [138–140].

In the early 2000s, Echtay et al. observed that protontransport by reconstituted UCP1 in liposomes is stronglystimulated by CoQ [53,141]; on the other hand, their ideaabout CoQ as an obligatory cofactor for uncoupling protonfunction besides FFAs was contested by other studies show-ing that UCP1 is similarly active in mitochondria isolatedfrom both CoQ-containing and -deficient transgenic yeaststrains [142]. Since then, new proposals were suggested forthe interaction of membranous CoQ molecules with FFA-acti-vated, PN-inhibited UCPs that mediate the proton conduct-ance in intact mitochondria [143]. Very recently, Woyda-Ploszczyca and Jarmuszkiewicz [144] have produced detailedexperimental evidence that the reduced form of CoQ acts as

a modulator of UCPs in Acanthamoeba castellanii mitochon-dria, showing that purine nucleotides exhibit an inhibitoryeffect over UCP-sustained uncoupling in the following de-scending order: GTP > ATP > GDP > ADP >> GMP > AMPand demonstrating that such inhibition is increased withdecreasing the CoQ reduction level, whereas it is alleviatedwith increasing ubiquinol concentration. To describe the ki-netic mechanism of this regulation, the same authors haveproposed a model in which the redox state of endogenousCoQ has no effect on the basal and FFA-induced UCP activityin the absence of PNs, but it affects the sensitivity to inhibi-tion by nucleotides because QH2 directly induces a competi-tive-like influence on the binding of PN (Fig. 6). These

results suggest that the low availability of respiratory sub-strates, corresponding to a low CoQ reduction level ( i.e., lowconcentration of ubiquinol), allows the inhibition of UCP1 byall guanine and adenine nucleotides, likely preserving ATPsynthesis yield. On the contrary, when the cytochrome path-way of the respiratory chain is impaired and the QH2 level isincreased, inhibition of UCPs is diminished to limit the pres-sure by the reducing equivalents ( i.e., NADH) and to preventthe formation of damaging ROS by mitochondria.

The structure of the PN-binding site is fairly well estab-lished for mammalian UCPs [145], but no attempts havebeen made to elucidate that of QH2. Although PNs and QH2,

which is more hydrophilic than oxidized CoQ, have very dif-ferent structures, some similarity could be found as theyboth are composed of aromatic rings. However, further stud-ies are necessary to determine whether PNs and QH2 couldbind to the same site in the protein.

It should also be emphasized that exogenous CoQ inits reduced form was shown to activate proton conductancethrough the production of superoxide, as superoxide dismu-tase inhibited such CoQ-induced uncoupling in kidney mito-chondria [146]. However, the current model for the activationof UCPs involving superoxide generated within mitochondriaat high membranous CoQ reduction level (as required for en-dogenous superoxide formation) implies the accumulation of lipid peroxidation products [147]. This phenomenon is, there-fore, regarded as an indirect, late response that can be im-portant whenever the formation of superoxide or the arisenendogenous CoQ redox state persists for prolonged timeperiods. On the contrary, the quick response of UCP activity

to rising concentrations of QH2 is provoked by QH2 itself,acting as a competitive regulator and directly relieving UCPinhibition from PNs [144].

8. Coenzyme Q as mitochondrialantioxidant and antiapoptotic agent

Besides being a redox component of the respiratory chain,physiological CoQ 10 behaves as a strong antioxidant in itsreduced form. Ubiquinol can interrupt the initiation of lipidperoxidation or also break the chain reaction by reacting

Fig. 6. A model for negative regulation of UCP purinenucleotide inhibition by QH2. At a given fatty acidconcentration, (A) at a low QH2 concentration, negativeregulation of PN-binding site does not occur and protonconductance through UCP is inhibited by purinenucleotides (PN). B: Conversely, an increased amount of QH2 could lead to decrease in the binding affinity of PN, thereby alleviating inhibition of UCP activity by thenucleotides. As a result, UCP-mediated H1 reuptake isenhanced. RC, respiratory chain. Image was taken withpermission from Woyda-Ploszczyca and Jarmuszkiewicz,Biochim. Biophys. Acta, 2011, 1807, 42–52, VC Elsevier.

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with lipid peroxide radicals generating ubisemiquinone anda nonradical lipid hydroperoxide [148]:

QH2 þ LOO ! Q þ LOOH þ Hþ:

Physiological ubisemiquinones having long side chains donot react with oxygen, except in complex III under thevery special conditions of the Q-cycle (see Section 5.3).On the other hand, short-chain ubiquinones, as CoQ 1 andalso hydroxydecyl-ubiquinone (idebenone), a compoundhaving clinical application, have a prooxidant effect incomplex I (see Section 6). Idebenone also inhibits com-plex I [149]; despite these effects, idebenone does notappear to be prooxidant in vivo ( cf . refs. 150 and 151),presumably because of prevalence of its reduced form inthe cells [152].

In mitochondria, the reduced antioxidant form of CoQ is regenerated by the respiratory chain. Besides the mito-chondrial respiratory chain, several enzymes catalyze CoQ reduction to achieve its antioxidant reduced state in eukary-

otic cells. NADH-cytochrome b5 reductase can reduceCoQ through a one-electron reaction mechanism [153]. Thesoluble enzyme NAD(P)H-quinone oxidoreductase 1 (NQO1,DT-diaphorase) can reduce quinones by a two-electron reac-tion and maintains the reduced state of CoQ 10 in vitro [154].A distinct cytosolic NADPH-CoQ reductase different fromNQO1 has also been described [155].

Murphy and coworkers [156] developed a new antiox-idant, named MitoQ, a ubiquinone derivative targeted tomitochondria by covalent attachment to a lipophilic triphe-nylphosphonium cation through an aliphatic carbon chain.Because of the large mitochondrial membrane potential,the cation was accumulated within mitochondria inside

cells, where the ubiquinone moiety inserted into the lipidbilayer and was reduced by the respiratory chain. Theubiquinol derivative thus formed was an effective antioxi-dant that prevented lipid peroxidation and protectedmitochondria from oxidative damage. After detoxifying areactive oxygen species (ROS), the ubiquinol moiety wasregenerated by the respiratory chain enabling its antioxi-dant activity to be recycled. In cell culture studies, themitochondrially localized antioxidant protected mamma-lian cells from hydrogen peroxide-induced apoptosis. Onthe other hand, MitoQs of different alkyl chain lengths,although specifically directed to mitochondria, are notable to restore electron transfer in CoQ-deficient

mitochondria [157] and do not always exhibit antioxidantproperties [158].

Other studies [159–161] have confirmed a protectiverole of CoQ 10 against apoptosis by showing inhibition of celldeath independently of its antioxidant effect, presumably byinhibition of opening of the PTP, a high-conductance proteinchannel located in the inner mitochondrial membrane [162],which depolarizes the mitochondrion and leads to therelease in the cytoplasm of proteins contained in the spacebetween the two mitochondrial membranes, such ascytochrome c and other factors that trigger the process of programed cell death (apoptosis).

In fact, quinones have been shown to exert a directeffect on PTP. Walter et al. [163] found that three functionalclasses of quinone analogs could be defined in relation toPTP: (i) PTP inhibitors, as CoQ 0, CoQ 2, and decylubiqui-none; (ii) PTP inducers, as idebenone (2,3-dimethoxy-5-methyl-6-(10-hydroxydecyl)-1,4-benzoquinone); and (iii)PTP-inactive quinones, which counteract the effects of bothinhibitors and inducers, such as CoQ 1. The quinones modu-late the PTP through a common binding site rather thanthrough oxidation-reduction reactions. Occupancy of thissite can modulate the PTP open-closed transitions, possi-bly through secondary changes of the Ca2þ-binding affinityfor the pore. PTP opening by its inducers led the cells toapoptosis [164].

In these studies, the effect of hydrophobic long-chainquinones could not be investigated; however, an indirectstudy [165] suggests out that CoQ 10 may be a PTP inhibitor.These authors exposed SHSY5Y neuroblastoma cells to neu-rotoxic b-amyloid peptides and oxygen-glucose deprivation(OGD) to investigate the neuroprotective effect of 10 lM

CoQ 10. Pore opening and superoxide anion concentrationwere increased in the Ab þ OGD group relative to controland were attenuated to the sham level when CoQ 10 wasadministered, indirectly demonstrating that CoQ 10 inhibitsthe opening of the pore besides reducing the concentrationof superoxide anion. Similar studies indicated a protectiveeffect of CoQ 10 on PTP opening in amitriptyline toxicity [166]and in ischemia and reperfusion in the heart [167]. It is notclear in these studies whether the effect of CoQ 10 on thetransition pore is a direct one or is mediated by the antioxi-dant effect.

8.1. Transcriptional effects of CoQ Several studies have revealed effects of CoQ 10 on geneexpression [168–173]. In theory, these effects might be medi-ated directly by, for example, interactions with a transcrip-tion factor. However, ROS are also potent inducers of geneexpression. H2O2 has been identified as an activator of theproinflammatory nuclear transcription factor NFjB [174]. Inview of the antioxidant properties of the reduced form of CoQ 10 and the effective enzymatic conversion of oxidizedCoQ 10 into its reduced form, Q 10 might mediate its observedanti-inflammatory effects via gene expression. Triggering of cells with lipopolysaccharide LPS induces downstream sig-

naling cascades of the transcription factor NFjB, which inturn leads to the induction of inflammatory genes; Q 10 down-regulates LPS-inducible genes in the monocytic cell lineTHP-1, presumably due to its antioxidant impact on geneexpression [175]. In another study [176], caloric restrictioninhibited age-related expression of five genes in heart andcerebellum; among dietary antioxidants, lipoic acid andCoQ 10 were as effective as caloric restriction in the cerebel-lum. Similar effects at the transcription level are promotedby the mitochondria-direct derivative Mito-Q [177]. Thus, it islikely that all effects of CoQ at the genetic level may bemediated by its antioxidant effect.



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9. A working hypothesis formitochondrial medicine: Viciouscircle of oxidative stress andsupercomplex disorganization

An overwhelming body of evidence accumulated in the lastdecades has demonstrated that mitochondria have a central

role in the etiology and pathogenesis of most major chronicdiseases and in aging itself ( cf. refs. 1 and 178–180); theinvolvement of mitochondria in disease, which has generatedthe term ‘‘mitochondrial medicine’’ [181], has been largelyascribed to their central role in production of ROS and tothe damaging effect of ROS on these organelles. In particu-lar, damage to mitochondrial DNA (mtDNA) would induce

alterations of the polypeptides encoded by mtDNA in the re-spiratory complexes, with consequent decrease of electrontransfer activity, leading to further production of ROS, andthus establishing a vicious circle of oxidative stress andenergetic decline [182]. This fall of mitochondrial energetic

capacity is considered to be the cause of aging and age-related degenerative diseases [180], although the picture isfurther complicated [183] by the complex interplay andcrosstalk with nuclear DNA and the rest of the cell [184].

In this scenario, it is easy to foresee a deep implication of supercomplex organization as a missing link between oxidativestress and energy failure [48]. It is tempting to speculate thatunder conditions of oxidative stress a dissociation of complex I–III aggregates occurs, with loss of facilitated electron channel-ing and resumption of the less efficient pool behavior of thefree ubiquinone molecules. In such regard, the quoted study onloss of supercomplex organization and of efficient electrontransfer in heart failure [56] is a clear indication of the deleteri-

ous consequence of lack of supramolecular organization.As predicted by Lenaz and Genova [48], dissociation of

supercomplexes might have further deleterious consequen-ces, such as disassembly of complexes I and III subunitsand loss of electron transfer and/or proton translocation; itcannot be excluded that the consequent alteration of elec-tron transfer may elicit further induction of ROS generation.The observation that complex III alterations prevent properassembly of complex I has, therefore, deep pathologicalimplications beyond the field of genetic mitochondrial cyto-pathies. Following this line of thought, the different suscepti-bility of different types of cells and tissues to ROS damagemay depend, among other reasons, on the extent and tight-

ness of supercomplex organization of their respiratorychains, which depend on their hand on phospholipids con-tent and composition of their mitochondrial membranes.These changes may have deep metabolic consequences, asdepicted in the scheme in Fig. 7. An initial enhanced ROSgeneration due to different possible reasons and originatingin different districts of the cell besides mitochondria ( cf. ref.185) would induce supercomplex disorganization eventuallyleading to possible decrease of complex I assembly; boththe lack of efficient electron channeling and the loss of complex I would decrease NAD-linked respiration and ATPsynthesis, and the defect in OXPHOS might then lead to

compensatory enhancement of glycolysis to overcome theenergetic deficiency.

9.1. Peroxidized phospholipids preventsupercomplex formationTwo roles of phospholipid have been distinguished: (i) a dis-persive solubilization effect that can be duplicated by appro-priate detergents and (ii) a catalytic effect that can be spe-cifically fulfilled only by cardiolipin [186–190]. Indeed, thereare yet two additional possible roles that may need to bemet, particularly in the case of complex I and complex III.These roles might be to provide a sufficiently lipophilic envi-ronment for the interaction of the lipophilic electron carrier,ubiquinone, and to participate in linking together compo-nents of the respiratory chain [1,48].

Fig. 7. Effect of supercomplex disorganization onmitochondrial function. Membrane phospholipidperoxidation and consequent loss of supercomplex organization may occur following an oxidative stressinduced by genetic changes (i.e., mitochondrial DNA mutations) or by exogenous factors (i.e., ischemia andreperfusion); the ensuing destabilization of complex Iresults in OXPHOS deficiency and further oxidative

stress. As a consequence of these changes, cells maybe forced to rely on glycolysis for energy production.The derangement of mitochondrial metabolism withenhanced ROS generation may have an additionaldeleterious consequence in an abnormal propensity tothe permeability transition by inappropriate opening of the mtPTP and would result in a catastrophic series of events ultimately leading to cell death.

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The phospholipids in closest vicinity to the protein sur-face, as well as those in the free bilayer, are actually highlymobile and free to exchange, but cardiolipin was indicated astightly bound being more likely buried within the protein com-plexes [191–193]. The absolute requirement of cardiolipin (CL)for the activity of cytochrome oxidase, complex I, and complex III [187] suggests that this phospholipid plays a crucial role inthe coupled electron transfer process, but recent results alsoseem to indicate that cardiolipin stabilizes respiratory chainsupercomplexes as well as the individual complexes. The avail-ability of a cardiolipin-lacking yeast mutant provided the oppor-tunity to demonstrate that mitochondrial membranes still con-tained the III2-IV2 supercomplex, but that it was significantlyless stable than supercomplexes in the parental strain. Otherphospholipids that are increased in the mutant, including phos-phatidyl ethanolamine and phosphatidyl glycerol, could notsubstitute for cardiolipin and could not prevent dissociation of supercomplexes, showing 90% of the individual homodimersof complexes III and IV not organized into supercomplex underBN-PAGE conditions [194,195].

In a different study, the stability and assembly of com-plex IV were found to be reduced in yeast cells lacking Taz1[196], the hortolog of human gene for Tafazzin, an acyl trans-ferase involved in the synthesis of mature tetralinoleyl cardio-lipin [197]; mutations of Tafazzin in humans result in Barthsyndrome, a cardioskeletal myopathy with neutropenia, charac-terized by respiratory chain dysfunction [198]; it was laterfound [199] that the cardiolipin defect in Barth syndromeresults in destabilization of the supercomplexes by weakeningthe interactions between respiratory complexes. Tafazzin isfound in the inner membrane in a complex includingATP synthase and the adenine nucleotide carrier; the absenceof this complex due to Taz1 mutations induces altered cristae

morphology [200]. In neutrophils, the supercomplex organiza-tion of the respiratory chain is disturbed by default, even inhealthy individuals. Further disturbance in Barth syndromepatients may lie at the basis of their neutropenia [201].

It is well documented that exposure of mitochondria toROS can affect the respiratory activity via oxidative damageof cardiolipin, which is required for the optimal functioningof the enzyme complexes [202–206]. We have demonstratedby flux control analysis that the maintenance of a I–III super-complex after reconstitution of a protein fraction enrichedwith complexes I and III (R4B) into phospholipid vesicles athigh protein to lipid ratios (see above) is abolished if lipidperoxidation is induced by 2,20-azobis-(2-amidinopropane)

dihydrochloride before reconstitution [47,207]. Evidently, thedistortion of the lipid bilayer induced by peroxidation andthe alteration of the tightly bound phospholipids determinedissociation of the supercomplex originally present in thelipid-poor preparation.

9.2. Supercomplex disassembly abolishes CoQ channeling and resumes less efficient poolbehaviorElectron channeling is bound to elicit rates above thosedictated by diffusion and collisions between randomly dis-

persed enzymes. Electron transfer between complex I andcomplex III (NADH cytochrome c reductase) exhibits rateshigher than those predicted by the pool equation in proteoli-posomes enriched in complexes I and III at a protein tophospholipid ratio of 1:1 (when kinetic testing according toflux control analysis indicates the presence of a supercom-plex I–III). However, at a ratio of 1:30 (when the supercom-plex is dissociated), the rate of NADH cytochrome c reduc-tase is exactly that predicted by the pool equation on thebasis of the individual activities of complex I and complex III; this is a demonstration that supercomplex formationindeed enhances the rate of electron transfer above thatoccurring via a ubiquinone pool in the membrane [47,207],and loss of supercomplex organization resumes less efficientpool activity of the quinone.

A study on human neutrophil mitochondria [201] clearlypoints out the consequences of lack of supercomplex organi-zation. In neutrophils, energy is mainly provided by a highglycolytic rate [208]; mitochondria are present but their res-piration with NAD-linked substrates is not apparently used

for ATP synthesis. The activity of individual complexes ispresent although rather low for complexes I, III, and IV; itwas shown that the individual complexes were present onlyin nonassembled form with complete absence of supercom-plex organization. On the other hand, the supercomplexesare present in the myeloid cell line HL-60, but during differ-entiation to neutrophil-like cells, HL-60 loose supercomplex organization with consequent lack of respiration.

9.3. Loss of supercomplexes prevents stability andassembly of individual complexesThe first chromatographic isolation of a complete respirasome

(I1III4IV4 ) from digitonin-solubilized membranes of Paracoccusdenitrificans indicated that complex I is stabilized by assemblyinto the NADH oxidase supercomplex because attempts to iso-late complex I from mutant strains lacking complexes III or IVled to the complete dissociation of complex I under theconditions of BN-PAGE. Reduced stability of complex I in thosemutant strains was also apparent from an almost completeloss of NADH-ubiquinone oxidoreductase activity when thesame protocol as for parental strain was applied [50].

Analysis of the state of supercomplexes in humanpatients with an isolated deficiency of single complexes[209] and in cultured cell models harboring cytochrome bmutations [210,211] also provided evidence that the forma-

tion of respirasomes is essential for the assembly/stabilityof complex I. Genetic alterations leading to a loss of com-plex III prevented respirasome formation and led to second-ary loss of complex I, therefore primary complex III assem-bly deficiencies presented as complex III/I defects.Conversely, complex III stability was not influenced by theabsence of complex I.

D’Aurelio et al. [211] studied mtDNA complementationin human cells by fusing two cell lines, one containing ahomoplasmic mutation in a subunit of respiratory chain com-plex IV, COX I, and the other a distinct homoplasmic muta-tion in a subunit of complex III, cytochrome b. Upon cell



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fusion, respiration was recovered in hybrids cells, indicatingthat mitochondria fuse and exchange genetic and proteinmaterials. The recovery of mitochondrial respiration corre-lated with the presence of supramolecular structures (super-complexes) containing complexes I, III and IV; criticalamounts of complex III or IV are therefore required in orderfor supercomplexes to form and provide mitochondrial func-tional complementation. From these findings, supercomplex assembly emerged as a necessary step for respiration, itsdefect setting the threshold for respiratory impairment inmtDNA mutant cells.

Several other studies in mutants and patients havingspecific defects in a single respiratory complex have sug-gested that complex III and to a lesser extent complex IVare involved in the assembly and stabilization of complex Iin mammals [199,212,213].

9.4. Disassembly of supercomplex organizationenhances generation of ROSAlthough no direct study is available on the effect of supra-

molecular organization on ROS production by the respiratorychain, indirect circumstantial evidence suggests that super-complex assembly may limit the extent of superoxide gener-ation by the respiratory chain.

It has been discussed in Section 6.2 that two potentialsites for oxygen reduction exist in complex I, represented byFMN and iron–sulfur cluster N2; controversial results fromdifferent laboratories working either on isolated complex I oron mitochondrial membranes generally indicate that N2 as asource of ROS would be predominant in membrane particles,whereas FMN might become available after complex I isola-tion. A reasonable hypothesis is that FMN becomes exposedto oxygen only when complex I is dissociated from complex III. Although the molecular structure of the individual com-plexes does not allow to envisage a close apposition of thematrix arm of complex I, where FMN is localized, with eithercomplex III or IV [30,31,33], the actual shape of the I 1III2IV1supercomplex from bovine heart [33] suggests a slightly dif-ferent conformation of complex I in the supercomplex with asmaller angle of the matrix arm with the membrane arm show-ing a higher bending toward the membrane (and presumablycomplex III), in line with the notion that complex I mayundergo important conformational changes [214]. Moreover,the observed destabilization of complex I in the absence of supercomplex (Section 9.3) may render the 51-kDa subunit

containing the FMN more ‘‘loose’’ allowing it to interact withoxygen. The elevated ROS production observed in P. anserinarespiring on AOX, where the major form of complex I is a I 2III2supercomplex rather than the usual I1III2 supercomplex [215],is in line with this reasoning, because it is likely that thecomplex I dimer may undergo a less tight interaction than acomplex I monomer with the complex III dimer.

9.5. Supercomplex loss accompanies pathologicalchangesThere are some observations in the literature showing thatpathological changes are accompanied by loss or decrease

of supercomplex organization of the respiratory chain. Insome instances, an enhanced ROS generation by mitochon-dria was observed under the same condition.

9.5.1. Aging. The ‘‘mitochondrial theory of aging’’ [216] isbased on the hypothesis that mitochondrial DNA somaticmutations, caused by accumulation of oxygen radicals dam-age, induce alterations of the OXPHOS machinery culminat-ing in an energetic failure that is at the basis of cellular se-nescence. Moreover, a vicious circle [182] can be establishedbecause the accumulated damage to the respiratory chainwould enhance ROS generation. Many reports (reviewed inref. 179) demonstrate that the rate of production of ROSfrom mitochondria increases with age in mammalian tissuesand in fibroblasts during replicative cell senescence, consid-ered to represent a plausible model of in vivo aging [217].Trifunovic et al. [218] showed that expression of a proofread-ing-deficient mtDNA polymerase in a homozygous knock-inmouse strain leads to increased levels of somatic mtDNA

mutations causing progressive respiratory chain deficiency;the mice develop symptoms strikingly r

genova_2011 new developments on the functions of coenzyme q in mitochondrial - critical review

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