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You are watching: What does the chemiosmotic hypothesis claim?

Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 2002.

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Conceptual Insights, Energy Transformations in OxidativePhosphorylation

View this media module for an animated, interactive summary of howelectron transfer potential is converted into proton-motive force and,finally, phosphoryl transfer potential in oxidativephosphorylation.



A molecular assembly in the inner mitochondrial membrane carries out the synthesis ofATP. This enzyme complex was originally called the mitochon-drialATPase or F1F0ATPase because it was discovered through its catalysis of thereverse reaction, the hydrolysis of ATP. ATP synthase, itspreferred name, emphasizes its actual role in the mitochondrion. It is also calledComplex V.

How is the oxidation of NADH coupled to the phosphorylation of ADP? It was firstsuggested that electron transfer leads to the formation of a covalent high-energyintermediate that serves as a high phosphoryl transfer potential compound or to theformation of an activated protein conformation, which then drives ATP synthesis. Thesearch for such intermediates for several decades proved fruitless.

In 1961, Peter Mitchell proposed that electron transport and ATP synthesis arecoupled by a proton gradient across the inner mitochondrialmembrane rather than by a covalent high-energy intermediate or anactivated protein conformation. In his model, the transfer of electrons through therespiratory chain leads to the pumping of protons from the matrix to the cytosolicside of the inner mitochondrial membrane. The H+ concentration becomeslower in the matrix, and an electrical field with the matrix side negative isgenerated (Figure 18.25). Mitchell"s idea,called the chemiosmotic hypothesis, was that this proton-motiveforce drives the synthesis of ATP by ATP synthase. Mitchell"s highly innovativehypothesis that oxidation and phosphorylation are coupled by a proton gradient isnow supported by a wealth of evidence. Indeed, electron transport does generate aproton gradient across the inner mitochondrial membrane. The pH outside is 1.4 unitslower than inside, and the membrane potential is 0.14 V, the outside being positive.As we calculated in Section 18.2.2, thismembrane potential corresponds to a free energy of 5.2 kcal (21.8 kJ) per mole ofprotons.


Figure 18.25

Chemiosmotic Hypothesis. Electron transfer through the respiratory chain leads to the pumping ofprotons from the matrix to the cytosolic side of the inner mitochondrialmembrane. The pH gradient and membrane potential constitute aproton-motive force (more...)

An artificial system was created to elegantly demonstrate the basic principle of thechemiosmotic hypothesis. Synthetic vesicles containing bacteriorhodopsin, apurple-membrane protein from halobacteria that pumps protons when illuminated, andmitochondrial ATP synthase purified from beef heart were created (Figure 18.26). When the vesicles were exposedto light, ATP was formed. This key experiment clearly showed that therespiratory chain and ATP synthase are biochemically separate systems, linkedonly by a proton-motive force.


Figure 18.26

Testing the Chemiosmotic Hypothesis. ATP is synthesized when reconstituted membrane vesicles containingbacteriorhodopsin (a light-driven proton pump) and ATP synthase areilluminated. The orientation of ATP synthase in this reconstitutedmembrane is (more...)

18.4.1. ATP Synthase Is Composed of a Proton-Conducting Unit and a CatalyticUnit

Biochemical, electron microscopic, and crystallographic studies of ATP synthasehave revealed many details of its structure (Figure 18.27). It is a large, complex membrane-embedded enzyme thatlooks like a ball on a stick. The 85-Å-diameter ball, called the F1subunit, protrudes into the mitochondrial matrix and contains the catalyticactivity of the synthase. In fact, isolated F1 subunits displayATPase activity. The F1 subunit consists of five types of polypeptidechains (α3, β3, γ, δ, and ϵ) with the indicatedstoichiometry. The α and β subunits, which make up the bulk of theF1, are arranged alternately in a hexameric ring; they are homologousto one another and are members of the P-loop NTPase family (Section 9.4.1). Both bind nucleotides but only the βsubunits participate directly in catalysis. The central stalk consists of twoproteins: γ and ϵ. The γ subunit includes a long α-helical coiled coil thatextends into the center of the α3β3 hexamer. The γsubunit breaks the symmetry of the α3β3hexamer: each of the β subunits is distinct by virtue of its interactionwith a different face of γ. Distinguishing the three β subunits iscrucial for the mechanism of ATP synthesis.

Figure 18.27

Structure of ATP Synthase. A schematic structure is shown along with detailedstructures of the components for which structures have beendetermined to high resolution. The P-loop NTPase domains of the αand β subunits are indicated (more...)

The F0 subunit is a hydrophobic segment that spans the innermitochondrial membrane. F0contains the proton channel of the complex. This channelconsists of a ring comprising from 10 to 14 c subunits that areembedded in the membrane. A single a subunit binds to the outsideof this ring. The proton channel depends on both the a subunit andthe c ring. The F0 and F1 subunits areconnected in two ways, by the central γϵ stalk and by an exterior column. Theexterior column consists of one a subunit, two bsubunits, and the δ subunit. As will be discussed shortly, we can think of theenzyme as consisting of two functional components: (1) a moving unit, orrotor, consisting of the c ring and the γϵstalk, and (2) a stationary unit, or stator, composed of theremainder of the molecule.

18.4.2. Proton Flow Through ATP Synthase Leads to the Release of Tightly Bound ATP:The Binding-Change Mechanism

Conceptual Insights, ATP Synthase as Motor Protein

looks further into the chemistry and mechanics of ATP synthaserotation.

The actual substrates are Mg2+ complexes of ADP and ATP, as in allknown phosphoryl transfer reactions with these nucleotides. A terminal oxygenatom of ADP attacks the phosphorus atom of Pi to form a pentacovalentintermediate, which then dissociates into ATP and H2O (Figure 18.28). The attacking oxygen atomof ADP and the departing oxygen atom of Pi occupy the apices of atrigonal bipyramid.

Figure 18.28

ATP Synthesis Mechanism. One of the oxygen atoms of ADP attacks the phosphorus atom ofPi to form a pentacovalent intermediate, which thenforms ATP and releases a molecule of H2O.

How does the flow of protons drive the synthesis of ATP? The results ofisotopic-exchange experiments unexpectedly revealed that enzyme-boundATP forms readily in the absence of a proton-motive force. When ADPand Pi were added to ATP synthase in H218O,18O became incorporated into Pi through the synthesisof ATP and its subsequent hydrolysis (Figure18.29). The rate of incorporation of 18O intoPi showed that about equal amounts of bound ATP and ADP are inequilibrium at the catalytic site, even in the absence of a proton gradient.However, ATP does not leave the catalytic site unless protons flow through theenzyme. Thus, the role of the proton gradient is not to form ATP but torelease it from the synthase.

Figure 18.29

ATP Forms Without a Proton-Motive Force But Is NotReleased. The results of isotope-exchange experiments indicate thatenzyme-bound ATP is formed from ADP and Pi in the absenceof a proton-motive force.

On the basis of these and other observations, Paul Boyer proposed abinding-change mechanism for proton-driven ATP synthesis.This proposal states that changes in the properties of the three β subunitsallow sequential ADP and Pi binding, ATP synthesis, and ATP release.The concepts of this initial proposal refined by more recent crystallographicand other data yield a satisfying mechanism for ATP synthesis. As already noted,interactions with the γ subunit make the three β subunits inequivalent (Figure 18.30). One β subunit can be in theT, or tight, conformation. This conformation binds ATP with great avidity.Indeed, its affinity for ATP is so high that it will convert bound ADP andPi into ATP with an equilibrium constant near 1, as indicated bythe aforediscussed isotopic-exchange experiments. However, the conformation ofthis subunit is sufficiently constrained that it cannot release ATP. A secondsubunit will then be in the L, or loose, conformation. This conformation bindsADP and Pi. It, too, is sufficiently constrained that it cannotrelease bound nucleotides. The final subunit will be in the O, or open, form.This form can exist with a bound nucleotide in a structure that is similar tothose of the T and L forms, but it can also convert to form a more openconformation and release a bound nucleotide (Figure 18.31). This structure, with one of the three β subunits inan open, nucleotide-free state, as well as one with one of the β subunits in anucleotide-bound O conformation, have been observed crystallographically.

Figure 18.30

ATP Synthase Nucleotide-Binding Sites Are Not Equivalent. The γ subunit passes through the center of theα3β3 hexamer and makes thenucleotide-binding sites in the β subunits distinct from oneanother.

Figure 18.31

ATP Release From the β subunit in the open form. Unlike the tight and loose forms, the open form of the β subunit canchange conformation sufficiently to release bound nucleotides.

The interconversion of these three forms can be driven by rotation of the γsubunit (Figure 18.32). Suppose the γsubunit is rotated 120 degrees in a counterclockwise direction (as viewed fromthe top). This rotation will change the subunit in the T conformation into the Oconformation, allowing the subunit to release the ATP that has been formedwithin it. The subunit in the L conformation will be converted into the Tconformation, allowing the transition of bound ADP + Pi into ATP.Finally, the subunit in the O conformation will be converted into the Lconformation, trapping the bound ADP and Pi so that they cannotescape. The binding of ADP and Pi to the subunit now in the Oconformation completes the cycle. This mechanism suggests that ATP can besynthesized by driving the rotation of the γ subunit in the appropriatedirection. Likewise, this mechanism suggests that the hydrolysis of ATP by theenzyme should drive the rotation of the γ subunit in the opposite direction.

Figure 18.32

Binding-Change Mechanism for ATP Synthase. The rotation of the γ subunit interconverts the three β subunits. Thesubunit in the T (tight) form, which contains newly synthesized ATPthat cannot be released, is converted into the O (open) (more...)

18.4.3. The World"s Smallest Molecular Motor: Rotational Catalysis

Is it possible to observe the proposed rotation directly? Elegant experimentswere performed with the use of a simple experimental system consisting of clonedα3β3γ subunits only (Figure 18.33). The β subunits were engineered to containamino-terminal polyhistidine tags, which have a high affinity for nickel ions.This property of the tags allowed the α3β3 assembly to beimmobilized on a glass surface that had been coated with nickel ions. The γsubunit was linked to a fluorescently labeled actin filament to provide a longsegment that could be observed under a fluorescence microscope. Remarkably, theaddition of ATP caused the actin filament to rotate unidirectionally in acounterclockwise direction. The γ subunit was rotating, being driven bythe hydrolysis of ATP. Thus, the catalytic activity of anindividual molecule could be observed. The counterclockwise rotation isconsistent with the predicted mechanism for hydrolysis because the molecule wasviewed from below relative to the view shown in Figure 18.32.

Figure 18.33

Direct Observation of ATP-Driven Rotation in ATP Synthase. The α3β3 hexamer of ATP synthase is fixed to asurface, with the γ subunit projecting upward and linked to afluorescently labeled actin filament. The addition and subsequenthydrolysis (more...)

More detailed analysis in the presence of lower concentrations of ATP revealedthat the γ subunit rotates in 120-degree increments, with each stepcorresponding to the hydrolysis of a single ATP molecule. In addition, from theresults obtained by varying the length of the actin filament and mea-suring therate of rotation, the enzyme appears to operate near 100% efficiency; that is,essentially all of the energy released by ATP hydrolysis is converted intorotational motion.

18.4.4. Proton Flow Around the c Ring Powers ATP Synthesis

The direct observation of rotary motion of the γ subunit is strong evidence forthe rotational mechanism for ATP synthesis. The last remaining question is: Howdoes proton flow through F0 drive the rotation of the γ subunit?Howard Berg and George Oster proposed an elegant mechanism that provides a clearanswer to this question. The mechanism depends on the structures of thea and c subunits of F0 (Figure 18.34). The structure of thec subunit was determined both by NMR methods and by x-raycrystallography. Each polypeptide chain forms a pair of α helices that span themembrane. An aspartic acid residue (Asp 61) is found in the middle of the secondhelix. When Asp 61 is in contact with the hydrophobic part of the membrane, theresidue must be in the neutral aspartic acid form, rather than in the charged,aspartate form. From 9 to 12 c subunits assemble into a symmetricmembrane-spanning ring. Although the structure of the a subunit hasnot yet been experimentally determined, a variety of evidence is consistent witha structure that includes two proton half-channels that do not span the membrane(see Figure 18.34). Thus, protons canpass into either of these channels, but they cannot move completely across themembrane. The a subunit directly abuts the ring comprising thec subunits, with each half-channel directly interacting withone c subunit.

Figure 18.34

Components of the Proton-Conducting Unit of ATP Synthase. The c subunit consists of two α helices that span themembrane. An aspartic acid residue in the second helix lies on thecenter of the membrane. The structure of the a subunithas not yet (more...)

With this structure in mind, we can see how a proton gradient can drive rotationof the c ring. Suppose that the Asp 61 residues of the twoc subunits that are in contact with a half-channel have givenup their protons so that they are in the charged aspartate form (Figure 18.35), which is possible becausethey are in relatively hydrophilic environments inside the half-channel. Thec ring cannot rotate in either direction, because such arotation would move a charged aspartate residue into the hydrophobic part of themembrane. A proton can move through either half-channel to protonate one of theaspartate residues. However, it is much more likely to pass through the channelthat is connected to the cytosolic side of the membrane because the protonconcentration is more than 25 times as high on this side as on the matrix side,owing to the action of the electron-transport-chain proteins. The entry ofprotons into the cytosolic half-channel is further facilitated by the membranepotential of +0.14 V (positive on the cytoplasmic side), which increases theconcentration of protons near the mouth of the cytosolic half-channel.If the aspartate residue is protonated to its neutral form,thecring can now rotate, but only in a clockwise direction. Such arotation moves the newly protonated aspartic acid residue into contact with themembrane, moves the charged aspartate residue from contact with the matrixhalf-channel to the cytosolic half-channel, and moves a different protonatedaspartic acid residue from contact with the membrane to the matrix half-channel.The proton can then dissociate from aspartic acid and move through thehalf-channel into the proton-poor matrix to restore the initial state. Thisdissociation is favored by the positive charge on a conserved arginine residue(Arg 210) in the a subunit. Thus, the difference in protonconcentration and potential on the two sides of the membrane leads todifferent probabilities of protonation through the two half-channels, whichyields directional rotational motion. Each proton moves through themembrane by riding around on the rotating c ring to exit throughthe matrix half-channel (Figure18.36).

Figure 18.35

Proton Motion Across the Membrane Drives Rotation of the CRing. A proton enters from the intermembrane space into the cytosolichalf-channel to neutralize the charge on an aspartate residue in ac subunit. With this charge neutralized, thec ring can (more...)

Figure 18.36

Proton Path Through the Membrane. Each proton enters the cytosolic half-channel, follows a completerotation of the c ring, and exits through the otherhalf-channel into the matrix.

The c ring is tightly linked to the γ and ϵ subunits. Thus, as thec ring turns, these subunits are turned inside theα3β3 hexamer unit of F1. The exteriorcolumn formed by the two b chains and the δ subunit prevent theα3β3 hexamer from rotating. Thus, theproton-gradient-driven rotation of the c ring drives the rotationof the γ subunit, which in turn promotes the synthesis of ATP through thebinding-change mechanism. Recall that the number of c subunits inthe c ring appears to range between 10 and 14. This number issignificant because it determines the number of protons that must be transportedto generate a molecule of ATP. Each 360-degree rotation of the γ subunit leadsto the synthesis and release of three molecules of ATP. Thus, if there are 10c subunits in the ring (as was observed in a crystal structureof yeast mitochondrial ATP synthase), each ATP generated requires the transportof 10/3 = 3.33 protons. For simplicity, we will assume that 3 protons must flowinto the matrix for each ATP formed, but we must keep in mind that the truevalue may differ.

18.4.5. ATP Synthase and G Proteins Have Several Common Features

The α and β subunits of ATP synthaseare members of the P-loop NTPase family of proteins. In Chapter 15, we learned that thesignaling properties of other members of this family, the G proteins, depend ontheir ability to bind nucleoside triphosphates and nucleoside diphosphates withgreat kinetic tenacity. They do not exchange nucleotides unless they arestimulated to do so by interaction with other proteins. The binding-changemechanism of ATP synthase is a variation on this theme. The three differentfaces of the γ subunit of ATP synthase interact with the P-loop regions of the βsubunits to favor the structures of either the NDP- or NTP-binding forms or tofacilitate nucleotide release. The conformational changes take place in anorderly way, driven by the rotation of the γ subunit.

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