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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 because that an animated, interactive review of howelectron deliver potential is converted right into proton-motive pressure and,finally, phosphoryl move potential in oxidativephosphorylation.
A molecule assembly in the within mitochondrial membrane carries out the synthesis ofATP. This enzyme complex was originally called the mitochon-drialATPase or F1F0ATPase since it was uncovered through its catalysis the thereverse reaction, the hydrolysis of ATP. ATP synthase, itspreferred name, emphasizes the actual function in the mitochondrion. That is likewise calledComplex V.
How is the oxidation that NADH coupled to the phosphorylation the ADP? It to be firstsuggested that electron transfer leads come the formation of a covalent high-energyintermediate that serves together a high phosphoryl transport potential link or to theformation that an activated protein conformation, which then drives ATP synthesis. Thesearch for such intermediates because that several decades proved fruitless.
In 1961, Peter Mitchell proposed that electron transport and ATP synthetic arecoupled by a proton gradient throughout the inner mitochondrialmembrane rather than by a covalent high-energy intermediate or anactivated protein conformation. In his model, the transport of electrons v therespiratory chain leads to the pumping of proton from the matrix to the cytosolicside that the inner mitochondrial membrane. The H+ concentration becomeslower in the matrix, and an electrical field with the procession side an unfavorable isgenerated (Figure 18.25). Mitchell"s idea,called the chemiosmotic hypothesis, was the this proton-motiveforce drives the synthesis of ATP through ATP synthase. Mitchell"s highly innovativehypothesis that oxidation and also phosphorylation space coupled through a proton gradient isnow sustained by a wealth of evidence. Indeed, electron carry does generate aproton gradient across the inside mitochondrial membrane. The pH external is 1.4 unitslower 보다 inside, and also the membrane potential is 0.14 V, the external being positive.As us calculated in ar 18.2.2, thismembrane potential coincides to a complimentary energy that 5.2 kcal (21.8 kJ) per mole ofprotons.
Figure 18.25
Chemiosmotic Hypothesis. Electron transfer through the respiratory tract chain leads to the pumping ofprotons indigenous the matrix to the cytosolic next of the inner mitochondrialmembrane. The pH gradient and membrane potential constitute aproton-motive force (more...)
An fabricated system was developed to elegantly demonstrate the straightforward principle that thechemiosmotic hypothesis. Fabricated vesicles containing bacteriorhodopsin, apurple-membrane protein native halobacteria the pumps protons once illuminated, andmitochondrial ATP synthase purified from beef love were developed (Figure 18.26). As soon as the vesicles were exposedto light, ATP was formed. This key experiment clearly showed that therespiratory chain and ATP synthase room biochemically separate systems, linkedonly by a proton-motive force.
Figure 18.26
Testing the Chemiosmotic Hypothesis. ATP is synthesized once reconstituted membrane engine containingbacteriorhodopsin (a light-driven proton pump) and ATP synthase areilluminated. The orientation the 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 also crystallographic studies of ATP synthasehave revealed many details that its framework (Figure 18.27). That is a large, complex membrane-embedded enzyme thatlooks like a ball on a stick. The 85-Å-diameter ball, called the F1subunit, protrudes right into the mitochondrial matrix and contains the catalyticactivity of the synthase. In fact, diverted F1 subunits displayATPase activity. The F1 subunit is composed of five types of polypeptidechains (α3, β3, γ, δ, and also ϵ) with the indicatedstoichiometry. The α and β subunits, which make up the mass of theF1, are arranged alternately in a hexameric ring; they are homologousto one another and are members that the P-loop NTPase family members (Section 9.4.1). Both bind nucleotides but only the βsubunits participate straight in catalysis. The main stalk is composed of twoproteins: γ and also ϵ. The γ subunit consists of a long α-helical coiled coil thatextends right into the facility of the α3β3 hexamer. The γsubunit breaks the the opposite of the α3β3hexamer: every of the β subunits is distinctive by virtue that its interactionwith a different challenge of γ. Differentiating the three β subunits iscrucial for the device of ATP synthesis.
Figure 18.27
Structure of ATP Synthase. A schematic structure is shown together with detailedstructures of the components for i m sorry structures have actually beendetermined come high resolution. The P-loop NTPase domains of the αand β subunits are shown (more...)
The F0 subunit is a hydrophobic segment that spans the innermitochondrial membrane. F0contains the proton channel that the complex. This channelconsists that a ring consisting of from 10 to 14 c subunits that areembedded in the membrane. A single a subunit binding 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, through the main γϵ stalk and by one exterior column. Theexterior column consists of one a subunit, two bsubunits, and also the δ subunit. As will certainly be questioned shortly, we have the right to think the theenzyme as consisting that two sensible components: (1) a moving unit, orrotor, consists of the c ring and also the γϵstalk, and (2) a stationary unit, or stator, created of theremainder the the molecule.
18.4.2. Proton flow Through ATP Synthase leads to the release of Tightly tied ATP:The Binding-Change Mechanism
Conceptual Insights, ATP Synthase together Motor Protein
looks further into the chemistry and mechanics of ATP synthaserotation.
The actual substrates space Mg2+ complexes the ADP and also ATP, as in allknown phosphoryl move reactions through these nucleotides. A terminal oxygenatom the ADP assaults the phosphorus atom of Pi to form a pentacovalentintermediate, which climate dissociates right into ATP and also H2O (Figure 18.28). The attacking oxygen atomof ADP and also the departing oxygen atom of Pi occupy the apices that atrigonal bipyramid.
Figure 18.28
ATP synthesis Mechanism. One of the oxygen atom of ADP attacks the phosphorus atom ofPi to kind a pentacovalent intermediate, i m sorry thenforms ATP and also releases a molecule the H2O.
How does the flow of protons journey the synthesis of ATP? The results ofisotopic-exchange experiments unexpectedly revealed that enzyme-boundATP creates readily in the absence of a proton-motive force. When ADPand Pi were included to ATP synthase in H218O,18O came to be incorporated into Pi v the synthesisof ATP and also its subsequent hydrolysis (Figure18.29). The price of incorporation of 18O intoPi verified that around equal quantities of bound ATP and also ADP space inequilibrium at the catalytic site, even in the lack of a proton gradient.However, ATP does not leave the catalytic website unless protons circulation through theenzyme. Thus, the function of the proton gradient is no to form ATP but torelease it from the synthase.
Figure 18.29
ATP develops Without a Proton-Motive Force but Is NotReleased. The results of isotope-exchange experiments indicate thatenzyme-bound ATP is developed from ADP and also Pi in the absenceof a proton-motive force.
On the communication of these and other observations, Paul Boyer propose abinding-change mechanism for proton-driven ATP synthesis.This proposal states that alters in the properties of the three β subunitsallow sequential ADP and also Pi binding, ATP synthesis, and also ATP release.The principles of this early stage proposal sleek by an ext recent crystallographicand other data productivity a satisfying device for ATP synthesis. As already noted,interactions through the γ subunit do the three β subunits inequivalent (Figure 18.30). One β subunit can be in theT, or tight, conformation. This conformation binding ATP with good avidity.Indeed, that is affinity because that ATP is for this reason high that it will transform bound ADP andPi into ATP through an equilibrium consistent near 1, as shown bythe aforediscussed isotopic-exchange experiments. However, the configuration ofthis subunit is saturated constrained that it cannot release ATP. A secondsubunit will then be in the L, or loose, conformation. This configuration bindsADP and Pi. It, too, is sufficiently constrained that it cannotrelease tied nucleotides. The last subunit will be in the O, or open, form.This type can exist with a tied nucleotide in a framework that is comparable tothose that the T and L forms, yet it can also convert to type a an ext openconformation and release a bound nucleotide (Figure 18.31). This structure, with among the three β subunits inan open, nucleotide-free state, and also one with among the β subunits in anucleotide-bound O conformation, have actually been it was observed crystallographically.
Figure 18.30
ATP Synthase Nucleotide-Binding Sites space Not Equivalent. The γ subunit passes v the center of theα3β3 hexamer and also makes thenucleotide-binding sites in the β subunits unique from oneanother.
Figure 18.31
ATP relax From the β subunit in the open form. Unlike the chop and loose forms, the open form of the β subunit canchange conformation sufficiently to release bound nucleotides.
The interconversion of this three creates can be propelled by rotation of the γsubunit (Figure 18.32). Mean the γsubunit is rotated 120 levels in a counterclockwise direction (as perceived fromthe top). This rotation will readjust the subunit in the T conformation into the Oconformation, allowing the subunit to release the ATP that has actually been formedwithin it. The subunit in the l conformation will certainly be converted right into the Tconformation, enabling the shift of bound ADP + Pi right into ATP.Finally, the subunit in the O conformation will certainly be converted into the Lconformation, trapping the bound ADP and Pi so the 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 control the rotation the the γ subunit in the appropriatedirection. Likewise, this mechanism argues that the hydrolysis of ATP through theenzyme must drive the rotation of the γ subunit in opposing direction.
Figure 18.32
Binding-Change mechanism for ATP Synthase. The rotation the the γ subunit interconverts the three β subunits. Thesubunit in the T (tight) form, which consists of newly synthesized ATPthat cannot be released, is converted right into the O (open) (more...)
18.4.3. The World"s the smallest Molecular Motor: Rotational Catalysis
Is it feasible to watch the proposed rotation directly? Elegant experimentswere performed with the use of a straightforward experimental mechanism consisting of clonedα3β3γ subunits only (Figure 18.33). The β subunits were engineered come containamino-terminal polyhistidine tags, which have a high affinity because that nickel ions.This property of the tags allowed the α3β3 assembly come beimmobilized ~ above a glass surface ar that had actually been coated through nickel ions. The γsubunit was attached to a fluorescently labeling actin filament to provide a longsegment that can be observed under a fluorescence microscope. Remarkably, theaddition of ATP resulted in the actin filament to revolve unidirectionally in acounterclockwise direction. The γ subunit was rotating, being driven bythe hydrolysis of ATP. Thus, the catalytic activity of anindividual molecule can be observed. The counterclockwise rotation isconsistent with the predicted device for hydrolysis since the molecule wasviewed from listed below relative to the view displayed in figure 18.32.
Figure 18.33
Direct monitoring of ATP-Driven Rotation in ATP Synthase. The α3β3 hexamer the ATP synthase is resolved to asurface, with the γ subunit projecting upward and also linked to afluorescently labeling actin filament. The addition and subsequenthydrolysis (more...)
More detailed evaluation in the existence of reduced concentrations the ATP revealedthat the γ subunit rotates in 120-degree increments, v each stepcorresponding to the hydrolysis of a single ATP molecule. In addition, indigenous theresults obtained by varying the length of the actin filament and also mea-suring therate that rotation, the enzyme appears to operate near 100% efficiency; the is,essentially all of the energy released by ATP hydrolysis is convert intorotational motion.
18.4.4. Proton Flow about the c Ring powers ATP Synthesis
The direct observation that rotary activity of the γ subunit is solid evidence forthe rotational mechanism for ATP synthesis. The critical remaining concern is: Howdoes proton circulation through F0 drive the rotation of the γ subunit?Howard Berg and also George Oster propose an elegant mechanism that provides a clearanswer to this question. The mechanism depends top top the structures of thea and c subunits that F0 (Figure 18.34). The structure of thec subunit was determined both by NMR methods and by x-raycrystallography. Each polypeptide chain develops a pair the α helices that expectations themembrane. An aspartic mountain residue (Asp 61) is uncovered in the middle of the secondhelix. As soon as Asp 61 is in contact with the hydrophobic part of the membrane, theresidue need to be in the neutral aspartic mountain form, quite than in the charged,aspartate form. From 9 to 12 c subunits assemble right into a symmetricmembrane-spanning ring. Although the structure of the a subunit hasnot yet been experimentally determined, a selection of evidence is continual witha framework that consists of two proton half-channels that perform not span the membrane(see figure 18.34). Thus, proton canpass right into either of this channels, however they can not move fully across themembrane. The a subunit directly abuts the ring consisting of thec subunits, v each half-channel directly interacting withone c subunit.
Figure 18.34
Components that the Proton-Conducting Unit of ATP Synthase. The c subunit consists of 2 α helices that expectancy themembrane. One aspartic acid residue in the 2nd helix lies on thecenter of the membrane. The framework of the a subunithas no yet (more...)
With this structure in mind, we deserve to see just how a proton gradient deserve to drive rotationof the c ring. Expect that the Asp 61 residual water of the twoc subunits that are in contact with a half-channel have actually givenup their protons so the they are in the charged aspartate kind (Figure 18.35), i m sorry is feasible becausethey space in relatively hydrophilic atmospheres inside the half-channel. Thec ring cannot revolve in either direction, due to the fact that such arotation would relocate a fee aspartate residue into the hydrophobic component of themembrane. A proton deserve to move with either half-channel come protonate one of theaspartate residues. However, the is much an ext likely to pass through the channelthat is linked to the cytosolic next of the membrane due to the fact that the protonconcentration is an ext than 25 times as high on this side as on the matrix side,owing to the activity of the electron-transport-chain proteins. The entrance ofprotons into the cytosolic half-channel is further facilitated by the membranepotential the +0.14 V (positive ~ above the cytoplasmic side), which increases theconcentration the protons near the mouth of the cytosolic half-channel.If the aspartate residue is protonated come its neutral form,thecring have the right to now rotate, but only in a clockwise direction. Such arotation moves the freshly protonated aspartic acid residue into call with themembrane, moves the fee aspartate residue from contact with the matrixhalf-channel to the cytosolic half-channel, and also moves a different protonatedaspartic acid residue from call with the membrane to the procession half-channel.The proton deserve to then dissociate native aspartic acid and also move v thehalf-channel into the proton-poor procession to regain the initial state. Thisdissociation is favored by the optimistic charge top top a conserved arginine residue(Arg 210) in the a subunit. Thus, the distinction in protonconcentration and potential on the two sides that the membrane leads todifferent probabilities that protonation through the two half-channels, whichyields directional rotational motion. Every proton moves with themembrane through riding roughly on the rotating c ring to exit throughthe procession half-channel (Figure18.36).
Figure 18.35
Proton Motion throughout the Membrane cd driver Rotation that the CRing. A proton enters indigenous the intermembrane space into the cytosolichalf-channel to neutralize the charge on an aspartate residue in ac subunit. V this charge neutralized, thec ring have the right to (more...)
Figure 18.36
Proton path Through the Membrane. Every proton enters the cytosolic half-channel, follows a completerotation of the c ring, and also exits through the otherhalf-channel into the matrix.
The c ring is tightly attached to the γ and ϵ subunits. Thus, as thec ring turns, these subunits room turned inside theα3β3 hexamer unit of F1. The exteriorcolumn developed by the 2 b chains and the δ subunit protect against theα3β3 hexamer from rotating. Thus, theproton-gradient-driven rotation the the c ring cd driver the rotationof the γ subunit, which subsequently promotes the synthesis of ATP v thebinding-change mechanism. Recall that the number of c subunits inthe c ring shows up to selection between 10 and also 14. This number issignificant since it determines the variety of protons that must be transportedto create a molecule of ATP. Every 360-degree rotation the the γ subunit leadsto the synthesis and also release of 3 molecules of ATP. Thus, if there are 10c subunits in the ring (as was observed in a decision structureof yeast mitochondrial ATP synthase), each ATP created requires the transportof 10/3 = 3.33 protons. Because that simplicity, we will certainly assume that 3 protons must flowinto the procession for every ATP formed, but we need to keep in mind the the truevalue might differ.
18.4.5. ATP Synthase and also G Proteins have actually Several usual Features
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