OPINION ARTICLE

Hypothesis of lipid-phase-continuity proton transfer for aerobicATP synthesisAlessandro M Morelli, Silvia Ravera, Daniela Calzia and Isabella Panfoli

The basic processes harvesting chemical energy for life are driven by proton (Hþ ) movements. These are accomplished by themitochondrial redox complex V, integral membrane supramolecular aggregates, whose structure has recently been described byadvanced studies. These did not identify classical aqueous pores. It was proposed that Hþ transfer for oxidative phosphorylation(OXPHOS) does not occur between aqueous sources and sinks, where an energy barrier would be insurmountable. This suggests anovel hypothesis for the proton transfer. A lipid-phase-continuity Hþ transfer is proposed in which Hþ are always bound tophospholipid heads and cardiolipin, according to Mitchell’s hypothesis of asymmetric vectorial Hþ diffusion. A phase separation isproposed among the proton flow, following an intramembrane pathway, and the ATP synthesis, occurring in the aqueous phase.This view reminiscent of Grotthus mechanism would better account for the distance among the Fo and F1 moieties of FoF1–ATPsynthase, for its mechanical coupling, as well as the necessity of a lipid membrane. A unique active role for lipids in the evolution oflife can be envisaged. Interestingly, this view would also be consistent with the evidence of an OXPHOS outside mitochondria alsofound in non-vesicular membranes, housing the redox complexes.

Journal of Cerebral Blood Flow & Metabolism advance online publication, 2 October 2013; doi:10.1038/jcbfm.2013.175

Keywords: ATP; cardiolipin; FoF1–ATP synthase; mitochondria; oxidative phosphorylation; proton transfer

INTRODUCTIONChemical energy for living matter is mostly supplied by theFoF1–ATP synthases (ATP synthases), multimeric proteins, whichemploy a rotary mechanism driven by proton-motive force. Inturn, protons (Hþ ) are translocated by electron transport chain(ETC). The structure and organization of ATP synthases, the‘splendid nanomolecular machine’1 that mechanically synthesizeATP from ADP and Pi are well described, but how its rotation isdriven by proton flow and how this energy is converted intocatalysis are less clear.2 F1 moiety is peculiar in that it is the onlyenzyme as yet known channeling energy to the reactant speciesby means of mechanical force. The topic of Hþ cycling, pivotal inoxidative phosphorylation, is central in energy conversion.3,4

Mitchell’s chemiosmotic theory foresaw the existence of anHþ translocation by the ETC, coupled to the transfer ofelectrons to oxygen with the formation of water, being theenergy involved in the process in the form of Hþ flux, convertedto ATP by FoF1–ATP synthase nanomotor.3 Hþ was also pivotal forthe origin of life. The ‘hydrogen hypothesis’ for the origin of thefirst eukaryote posits that an anaerobic hydrogen-dependentnucleus-bearing archaebacterium host phagocytized an a-proteo-bacterium (that would become a mitochondrion) with an initialbenefit represented by molecular hydrogen production by theendosymbiont, thus challenging the traditional view according towhich ATP was the reward.5

In his last paper, Mitchell gives us a clue: Hþ movements arevectorial, not scalar. ‘In chemiosmotic systems, the pathways ofspecific ligand conduction are spatially orientated throughosmoenzymes and porters in which the actions of chemical

group, electron and solute transfer occur as vectorial (or highertensorial order) diffusion processes down gradients of totalpotential energy that represent real spatially directed fields offorce’.6 Some uncertainty exists about the nano-local pathway ofHþ in a respiring membrane, long debated.7–9 Also, it has recentlybecome clear that in the respiring membranes, the ATP synthasesform supramolecular complexes,10 with ETC proteins and lipidssuch as cardiolipin, central to their functioning.2

Proton Handling Inside Respiring MembranesHþ do not exist as free species in the aqueous bulk, where theyform hydronium ion, with a desolvation cost of more than500 meV.11 On the other hand, Hþ would not be able to freelyreside inside the lipid bilayer, as they possess the highest charge/mass ratio than any ion. Moreover, a potential barrier representedby ordered water molecules on the surface of biological mem-branes would prevent Hþ to diffuse.12 How can enoughHþ motive force be generated across inner mitochondrial mem-branes to synthesize ATP? Perhaps, this is a general open questionfor ATP synthase, as recently pointed out,2 and the crux of thequestion. The current concept according to which the rotation ofthe rotor is fueled by Hþ translocation in aqueous phase continuitymay be inadequate. Starting from the newly acquired structuraldata on ETC I;13,14 ETC III;15 and ATP synthase,16 some light is shedon the finely integrated processes of Hþ translocation by ETClinked to oxygen reduction, and nanomechanical ATP synthesis.

A new criterion for Hþ transfer inside respiring membranescan be envisaged. Evidence on the buffering capacity of the

Department of Pharmacy-Biochemistry Lab, University of Genova, Genova, Italy. Correspondence: Professor I Panfoli, University of Genova, School of Medical and PharmaceuticalSciences, DIFAR-Department of Pharmacy, Biochemistry Lab. Viale Benedetto XV, 3 16132 Genova, Italy.E-mail: Isabella.Panfoli@unige.itThe work was supported by grants from Compagnia di San Paolo, for the Neuroscience Program for the research project entitled ‘Energetic metabolism in myelinated axon:a new trophic role of myelin sheath’.Received 21 March 2013; revised 9 September 2013; accepted 12 September 2013

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phospholipid head groups has been gathered.9,17 In his paperpresented by Lehninger, Haines said: ‘anionic lipid head groups inbiological membranes share protons as acid-anion dimers andanionic lipids thus trap and conduct protons along the head groupdomain of bilayers that contain such anionic lipids. Protonspumped from the other side of the membrane may enter andmove within the head group sheet because the protonation rateof negatively charged proton acceptors is five orders ofmagnitude faster than that of water’.9 A ‘lateral Hþ delivery’ i.e.the transfer of Hþ along the surface would predominate overtransfer between aqueous sources and sinks.11 Hþ migratingthrough acid carriers is in interesting accordance to the structuraldiffusion mechanism proposed by Theodor von Grotthus(structural diffusion of Hþ ) over 2 centuries ago.18 Lehningerstated that: ‘..the Hþ movements occurring during electrontransporty do not necessarily mean that such Hþ movementsalso occur between the two bulk phases (the matrix and medium)during the actual normal process of oxidative phosphorylation–translocation of Hþ between two bulk phases may not bea necessary event during oxidative phosphorylation; rather,it is possible that charge movements within the membrane arethe fundamental processes serving as the vehicle of energytransductiony.’.19

When there is the actual need for acidification of the medium,i.e. the transfer of Hþ in aqueous phase continuity, proteins thatbelong to a different class with respect to FoF1–ATP synthase areinvolved. Notably, biological systems typically use the sameprocess for the same purpose. These are P-type ATP-driven cationtransporters such as Hþ /Kþ -ATPase. The kidney tubular20 andgastric H, K-ATPase21 exchange ions (Hþ , chloride ions, andpotassium ions) across the cell membrane, extruding Hþ througha channel.21

Lipid-Phase-Continuity Hþ TransferIf Hþ always reside inside the proteolipid phase of the membrane,a situation already depicted in 1961,22 the process of ATPsynthesis occurs with a net phase separation: Hþ would flowalong an intramembrane pathway, whereas ATP synthesis wouldbe in the aqueous phase. In fact, subunit F1 of ATP synthase isabout 10 nm away from the inner mitochondrial membrane (IMM)surface. Such phase separation in turn sets the need for a couplingof the two processes, which is in fact the case. A nanomechanicalcoupling appears a good solution. Interestingly, it was recentlyproposed also for ETC I.14

A Hþ circuit would be established inside respiring membranes.Negative charges of phosphate groups would lie on both sides ofthe IMM, whereas positive ones would reside at the nonpolarcenter of the membrane, where the hydrocarbon tails arecompact. Such possibility has been depicted before.23 Thepossible involvement of the supercomplexes, a supramoleculararrangement of the OXPHOS complexes in the IMM, should alsobe considered.10 These supercomplexes might funnel Hþ , alsoburying cytochrome c. The proteolipid phase appears ideal forhydrophobic compound chemical reactions. Mulkidjanianproposed the existence of ‘shallow DpH- and Dc-sensitiveproton traps, mechanistically linked to the functional groups inthe membrane interior’.24 An active involvement of plasmamembrane environment was recently described in bacteria,mitochondria ancestors, for allocation of diacylglycerol substrateof diacylglycerol kinase.25 This sheds light on the peculiar catalyticmechanisms of membrane enzymes. It is noteworthy that thesedata come from X-ray studies on lipid mesophase crystal.26

The possibility that Hþ are confined to the Helmholtz layer ofthe IMM, where they diffuse faster was also proposed by Kell,7 andseems confirmed by the recent pivotal advances in the knowledgeof the detailed structure of some ETC and ATP synthase,13,14,16

which did not prove the existence of a aqueous pores. Instead, the

structure of ETC I revealed a coupling mechanism involvingantiporter-like subunits acting at distance from the interface withthe hydrophilic domain.14 The proteolipid environment was saidto thermodynamically stabilize the protonation state of the Fo

rotor.16 It may be hypothesized that uncouplers (such as classical2,4 dinitrophenol) transfer Hþ from the phospholipids to the ETCat the center of the membrane.

The Role of CardiolipinA role in Hþ on guidance from the ETC to the phosphates is likelyplayed by cardiolipin (CL). Cardiolipin is inextricably linked to theoperation of the OXPHOS proteins,27 due to its unique ability toact as a Hþ -trap.28 We may speculate that CL, a lipid moleculecontaining two hydrophobic domains, acts as a Hþ shuttle at thecentr of the membrane between subunit a of Fo and ETC astentatively depicted in Figure 1. Panel A illustrates the possibilitythat the two lipid domains of cardiolipin (CL) molecule can beeither in a ‘closed’ form, when the phosphates are negativelycharged, in one leaflet (probably the periplasmic one29,30) of theIMM, with the two hydrophobic domains close together,consistent with what reported for complex III analyzed by X-raydiffraction,31 or in an ‘open’ conformation when CL accepts Hþ , inconditions favouring respiration and ATP synthesis. In the lattercase, the central part of CL loses polarity and the two lipiddiglyceride residue domains would be redistributed inside the twoleaflets of the IMM. Therefore, CL would be the only lipid that canbe arranged simultaneously in the two layers of the IMM. This isconsistent with the data on CL asymmetrical distribution,32 on theexistence of multiple binding sites for CL with ETC III,33 and on thefluorescent dye 10-N-nonyl acridine orange.34,35 In a quantitativeassays of CL, authors35 report that 10-N-nonyl acridine orange–cardiolipin fluorescence intensity is maximal in respiratory state 3during active respiration (i.e.: during active Hþ shuttling),decreases in respiratory state 4, reaching a minimum in non-respiring mitochondria. Interestingly, it was reported that 57% oftotal CL was present in the outer leaflets of inner membranes ofisolated mitochondria.30 Figure 1B, based on the knowledge of itsstructure,36,37 proposes that subunit a of ATP synthase transfersHþ to Glu 58 at the center of subunit c. Then Hþ through an Argresidue (R210 in E. coli38) would pass to respiratory complexes,which, in turn, transfer them to the periplasmic side of the IMM.This putative pathway would be in line with the charged aminoacids in subunit a.36,38 It may also be hypothesized thatprotonated CL stably realizes a kind of Hþ tunneling to thecenter of the IMM from the center of Fo moiety to the center ofthe ETC, which translocate Hþ to the periplasmic side.

Crystallography studies on cytochrome bc1 complex31 showedthat Hþ are acquired by CL, co-crystallized with the complex atthe center of the IMM. Cardiolipin lies at the center of complex III,involved in Hþ transfer.31 The detailed analysis of ATP synthasecarried out at the atomic level16 locates the Hþ transfer in thecentral area of the membrane. A direct interaction between ATPsynthase and CL has been demonstrated.37 Cardiolipin has alsobeen found on the so-called mitochondria-associatedmembranes.39 The lack of adequate conceptual chemical–physical tools to examine such processes must be remarked.Indeed, the intrinsic chemical propriety of proteolipid phases ofthe biological membranes is largely unknown. In fact, the theoryof acid/base conversion is referred to water phase. Also, due to theminimal inherent mass of Hþ , quantum mechanics (change ofmass with velocity) may be invoked for Hþ movements,reminiscent of the ‘uncertainty principle’ proposed by Szent-Gyorgyi for biological systems.40

Hypothesis of an Intramembrane PotentialThe asymmetric charge distribution would generate an intramem-brane potential, to fuel the rotation of the ATP synthase. The

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concept may be introduced of a ‘ Hþ pressure’ generated by theETC located inside the proteolipid phase of the IMM, mechanicallyconverted into ATP synthesis. So Fo can be fueled by both a Hþ

potential applied to the membrane, like in in vitro experi-ments,41,42 and a ‘Hþ pressure’, exclusive of the naturalrespiring membranes. The inconsistence of a membrane potential-driven backflow of Hþ ions has been already discussed.43 Thiswould be in accordance to Mitchell’s idea of a conceptualasymmetry of metabolism, which would be better described interms of vectorial forces rather than ‘conventional scalarenergies’.6 Measurements of mitochondrial IMM voltage gradientfound a positive charge on the matrix face,44 conflicting with thetheoretical polarity needed for Hþ translocation toward mito-chondrial matrix. Such measurements deserve revaluationbecause they agree with theoretical calculations.45,46

Notably, a Hþ cycle for OXPHOS confined in the proteolipidphase is in accordance to the coupling of the ETC with oxygenconsumption and ATP synthesis. If Hþ were translocated to andfrom the aqueous phase, the ETC would not stop acidifying theextramitochondrial milieu. Instead, it is known that only additionof ADP and Pi (a classical experimental procedure) can unload the‘Hþ pressure’. The concept of a collective Hþ -tunneling had beenalso proposed by Bartl et al47 in an FT-IR spectroscopy study of theFo complex of ATP synthase embedded into CL liposomes. Authorsreport that: ‘ya proton pathway is present in native Fo, in whichthe protons are shifted in a hydrogen-bonded chain with largeproton polarizability...for collective proton tunneling. Suchpathways are very efficient, because they conduct protonswithin picoseconds’.47 The presence of ‘sequestered domains inwhich Hþ are held in a metastable state out of equilibrium with

Figure 1. Tentative Hþ circuit inside respiring inner mitochondrial membrane (IMM). Panel A proposes that cardiolipin (CL) can exist in twoconformations: ‘closed’, with the two hydrophobic domains close together, when the phosphate residues are deprotonated (anionic) and theirpolarity prevails so that they lay close to the aqueous milieu; and ‘open’ (about 4.8 nm long) with the two hydrophobic domains laying in each ofthe two leaflets of the IMM, when the phosphate residues are protonated and become less polar. Panel B proposes that the Hþ (black dotted line)are transferred to the Glu 58 (E58) at the center of subunit c through a subunit of ATP synthase by proton tunneling. Hþ would flow from theperiplasmic side, always bound to phospholipid heads. This putative pathway would be in line with the existing charged amino acids in a subunit.Then, after almost one complete turn, Hþ would be acquired by an Arg residue and eventually by CL, generating a protonated bis-glycerolphosphate with negligible polarity. This can be arranged in each layer of the membrane. Hþ would be shuttled by CL towards the center of eachelectron transport chain (ETC) (generic respiratory complex, RC in Figure), which in turn transfers them to the periplasmic side of the membrane.

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those in the inner–outer aqueous bulk phases48’ has also beenreported for the thylakoid membranes.48

CONCLUSIONS AND PERSPECTIVESMitchell’s chemiosmotic theory3 allowed a huge step forward inthe field of bioenergetics. It has been said that:‘.. (Mitchell’s)experimental and conceptual work proved extraordinarilysuccessful in providing proof of the general hypothesis andprompting mechanisms of key components..’.4 It is worth pointingout that the model proposed here does not conflict with Mitchell’stheory. Rather, it reveals the still unrecognized potential ofMitchell’s theory, also in being extendable to the emergingawareness of extramitochondrial OXPHOS.49 New roles emerge forATP synthases, some of which were recently shown to beectopically expressed in many cellular membranes where theywould conduct extramitochondrial OXPHOS (HUVEC cells,50 rodouter segments,51,52 rat hepatocytes,53 isolated myelin,54 andcancer cells.38 In particular, the present hypothesis is consistentwith the reported possibility to conduct OXPHOS on a plasmamembrane, i.e. in the absence of a closed vesicle.38,53 Theproposed proteolipidic Hþ translocation would help understandextramitochondrial ATP synthases, acting without any trans-membrane Hþ gradient.

The hypothesis that myelin could act like a mitochondrion tocontribute to the energetic metabolism of neurons has beencriticized by Harris and Attwell.55 The problem was posed that anarrangement of the OXPHOS proteins is inconsistent with theexperimentally measured aerobic ATP synthesis. It was observedthat in myelin, the ATP synthase would function in reverse,hydrolyzing rather than synthesizing the ATP.55 It should be noted,however, that the ATP synthase molecular motor is driven by anHþ transfer chain (even if, the mechanism of proton-motiveconversion into a mechanical force driving the Fo rotor is largelyunknown). Therefore, conversely, a reverse motion of the motorwould generate an unlikely backward Hþ transfer.

So far, there is no direct evidence for the assumptions we made.Nevertheless, the hypotheses of phase separation and exclusion ofaqueous phase continuity for Hþ transfer allow to conceive anaerobic ATP synthesis occurring independently of closed compart-ments where to accumulate Hþ is not necessary for ATP synthesis,as also proposed for IMM by Lehninger half a century ago.56

A vectorial transfer of Hþ intrinsic to the IMM following theGrotthus mechanism, with a fine integration of Hþ translocationand electron transfer for direct coupling of ETC and ATP synthase,thanks to the lipid environment, underlines the unique role ofbiological membranes in the evolution of life. These would notonly have provided a permeability barrier and compartmentation,but had a fundamental role in arranging the proteins involved ingaining chemical energy for life.

DISCLOSURE/CONFLICT OF INTERESTThe authors declare no conflict of interest.

ACKNOWLEDGMENTSWe thank Giorgio Lenaz (University of Bologna) for his invaluable contribution andJulia J Harris and David Atwell (University College London) for stimulating this basicdiscussion.

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