Lecture 3 Oxidative Phosphorylation Metabolism III

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Metabolism IIIMetabolism III ± ±OxidativeOxidative phosphorylationphosphorylation

March 2, 2009



Essence of Oxidative Phosphorylation. Oxidation and ATP synthesis are coupled bytransmembrane proton fluxes.

Oxidative phosphorylation is the process in which ATP is formed as a result of the transfer

of electrons from NADH or FADH 2 to O 2 by a series of electron carriers.

Oxidative Phosphorylation



Cytosolic side or P-side

Matrix side or N-side

Role of mitochondria

� M itochondria contain the respiratory assembly, the enzymes of the citric acid cycle, and

the enzymes of fatty acid oxidation.

� Mitochondria has two membranes.

� Oxidative phosphorylation takes place in the inner mitochondrial membrane as opposed tomost of the reactions of TCA cycle that takes place in the Matrix.� The outer membrane is quite permeable to most small molecules and ions because itcontains many copies of mitochondrial porin, a 30±35 kd poreforming protein also known asVDAC, for voltage-dependent anion channel.� The two faces of this membrane will be referred to as the matrix side and the cytosolic side

(the latter because it is freely accessible to most small molecules in the cytosol). They are

also called the N and P sides, respectively, because the membrane potential is negative on thmatrix side and positive on the cytosolic side.



T he concept of Redox potential

If the reaction proceeds in the direction

the reactions in the half-cells (referred to

as half-reactions or couples) must be

Thus, electrons flow from the sample half-cell tothe standard reference half-cell, and the sample-

cell electrode is taken to be negative with respectto the standard-cell electrode. The reduction

potential of the X:X -couple is then observed

voltage at the start of the experiment (when X, X-,and H+ are 1 M). The reduction potential of the

H +:H 2 couple is defined to be 0 volts.

The meaning of the reduction potential is now evident. A negative reduction potential meansthat the reduced form of a substance has lower affinity for electrons than does H2, as in thepreceding example. A positive reduction potential means that the reduced form of asubstance has higher affinity for electrons than does H2. These comparisons refer tostandard conditions²namely, 1 M oxidant, 1 M reductant, 1 M H+, and 1 atmosphere H2. Thus,a strong reducing agent (such as NADH) is poised to donate electrons and has a negative

reduction potential, whereas a strong oxidizing agent (such as O 2 ) is ready to accept electrons and has a positive reduction potential.



The reduction potentials of many biologically important redox couples are known. It is

like those presented in chemistry texts except that a hydrogen ion concentration of 10-7

M (pH 7) instead of 1 M (pH 0) is the standard state adopted by biochemists. This

difference is denoted by the prime in E´ 0. Recall that the prime in G°´ denotes a

standard free-energy change at pH 7.The standard free-energy change G°´ is related to the change in reduction potential

E´ 0 by

in which n is the number of electrons transferred, F is a proportionality constant called

the faraday [23.06 kcal mol-1 V-1 (96.48 kJ mol-1 V-1)], E´ 0 is in volts, and G°´ is in

kilocalories or kilojoules per mole.

How can the energy associated with a proton gradient be quantified? The free-energychange for a species moving from one side of a membrane where it is at concentration c 1to the other side where it is at a concentration c 2 is given by in which Z is the electricalcharge of the transported species and V is the potential in volts across the membrane.

Under typical conditions for the inner mitochondrial membrane, the pH outside is 1.4units lower than inside [corresponding to log10 (c 2 /c 1) of 1.4] and the membrane potentialis 0.14 V, the outside being positive. Because Z = +1 for protons, the free-energy changeis (2.303 × 1.98 × 10-3 kcal mol-1 K-1 × 310 K × 1.4) + ( + 1 × 23.06 kcal mol-1 V-1 × 0.14 V) =

5.2 kcal mol-1 (21.8 kJ mol-1). Thus, each proton that is transportedout of the matrix to the cytosolic side corresponds to 5.2

kcal mol-1 of free energy.



Electron transport chain



Structure of NADH-Q Oxidoreductase (Complex I). The structure, determined byelectron microscopy at 22-Å resolution, consists of a membrane-spanning part and a longarm that extends into the matrix. NADH is oxidized in the arm, and the electrons aretransferred to reduce Q in the membrane.

Structure of NADH-Q Oxidoreductase

Matrix side

Cytosol side



How is AT P synthesis coupled to Proton gradient?



Chemiosmotic hypothesis

How is the oxidation of NADH coupled to the phosphorylation of ADP? It was first

suggested 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.

Testing the Chemiosmotic Hypothesis of

Peter Mitchell (1961). ATP is synthesizedwhen reconstituted membrane vesiclescontaining bacteriorhodopsin (a light-drivenproton pump) and ATP synthase areilluminated. The orientation of ATP synthase inthis reconstituted membrane is the reverse of that in the mitochondrion.



T he structure of F0 F1 AT Pase

F0, 10-14 c subunits, Proton conduit.

F1, (33) contains the ATP synthase activity(even in isolation)

Connectors, i> stalk in the inside.ii> Two b subunits connect a and on

the outside.

we can think of the enzyme as consisting of twofunctional components: (1) a moving unit, or rotor,

consisting of the c ring and the stalk, and (2) astationary unit, or stator, composed of the remainder of the molecule.



No. 161



Rotation of the c-complex

Proton Motion Across the Membrane Drives Rotation of the C Ring. A proton enters fromthe intermembrane space into the cytosolic half-channel to neutralize the charge on anaspartate residue in a c subunit. With this charge neutralized, the c ring can rotateclockwise by one c subunit, moving an aspartic acid residue out of the membrane into the

matrix half-channel. This proton can move into the matrix, resetting the system to its initialstate.



Proton Path Through the Membrane. Each proton

enters the cytosolic half-channel, follows a

complete rotation of the c ring, and exits through

the other half-channel into the matrix.

Thus, the difference in proton

concentration and potential on

the two sides of the membraneleads to different probabilities of

protonation through the two half-

channels, which yields

directional rotational motion.

Each proton moves through the

membrane by riding around onthe rotating c ring to exit throughthe matrix half-channel

Each 360-degree rotation of the subunit leads to the synthesis and

release of three molecules of ATP.



Glycerol 3-Phosphate Shuttle. Electrons from NADH can enter the mitochondrial electrontransport chain by being used to reduce dihydroxyacetone phosphate to glycerol 3-phosphate.Glycerol 3-phosphate is reoxidized by electron transfer to an FAD prosthetic

group in a membrane-bound glycerol 3-phosphate dehydrogenase. Subsequent electrontransfer to Q to form QH2 allows these electrons to enter the electron-transport chain.

Paying the cytosolic NADH debt



W hen cytosolic NADH transported by the

glycerol 3-phosphate shuttle is oxidized by therespiratory chain, 1.5 rather than 2.5 ATP are

formed. The yield is lower because FAD rather than NAD+ is the electron acceptor inmitochondrial glycerol 3-phosphatedehydrogenase. The use of FAD enableselectrons from cytosolic NADH to be

transported into mitochondria against an NADHconcentration gradient. The price of thistransport is one molecule of ATP per twoelectrons.


This glycerol 3-phosphate shuttle is especially prominent in muscle and

enables it to sustain a very high rate of oxidative phosphorylation. Indeed,some insects lack lactate dehydrogenase and are completely dependent onthe glycerol 3-phosphate shuttle for the regeneration of cytosolic NAD+.




Malate-Aspartate Shuttle. In the heart and liver, electrons from cytosolic NADH are brought into mitochondria

by the malate-aspartate shuttle, which is mediated by two membrane carriers and four enzymes. Electrons

are transferred from NADH in the cytosol to oxaloacetate, forming malate, which traverses the inner mitochondrial membrane and is then reoxidized by NAD+ in the matrix to form NADH in a reaction catalyzed

by the citric acid cycle enzyme malate dehydrogenase. The resulting oxaloacetate does not readily cross the

inner mitochondrial membrane, and so a transamination reaction is needed to form aspartate, which can be

transported to the cytosolic side. Mitochondrial glutamate donates an amino group, forming aspartate and -

ketoglutarate. In the cytoplasm, aspartate is then deaminated to form oxaloacetate and the cycle is restarted.

This shuttle, in contrast with the glycerol 3-phosphate shuttle, is readily reversible. Consequently,NADH can be brought into mitochondria by the malate- aspartate shuttle only if the NADH/NAD+ ratio

is higher in the cytosol than in the mitochondrial matrix. This versatile shuttle also facilitates the exchangeof key intermediates between mitochondria and the cytosol.



Mechanism of Mitochondrial ATP-ADP Translocase. The translocase catalyzes the coupledentry of ADP and exit of ATP into and from the matrix. The reaction cycle is driven by

membrane potential. The actual conformational change corresponding to eversion of thebinding site could be quite small.

T he Entry of ADP into Mitochondria Is Coupled to the Exitof AT P by AT P-ADP T ranslocase



In the presence of a positive membrane potential (as would be the case for an actively respiring

mitochondrion), the rate of binding-site eversion from the matrix to the cytosolic side is more

rapid for ATP than for ADP because ATP has one more negative charge. Hence, ATP is transported

out of the matrix about 30 times as rapidly as is ADP, which leads to a higher phosphoryl transfer

potential on the cytosolic side than on the matrix side. The translocase does not evert at an

appreciable rate unless a molecule of ADP is bound at the open, cytosolic site, which then everts

to the mitochondrial matrix side. This feature ensures that the entry of ADP into the matrix is

precisely coupled to the exit of ATP. The other side of the coin is that the membrane potential and

hence the proton-motive force are decreased by the exchange of ATP for ADP, which results in a

net transfer of one negative charge out of the matrix. ATP-ADP exchange is energetically

expensive; about a quarter of the energy yield from electron transfer by the respiratory chain is

consumed to regenerate the membrane potential that is tapped by this exchange process. The

inhibition of this process leads to the subsequent inhibition of cellular respiration as well



The phosphate carrier, which works in concert with ATP-ADP translocase, mediates the electroneutral exchange of

H2PO4- for OH- (or, indistinguishably, the electroneutral symport of H2PO4

- a nd H+). The combined actio n o f t hese

t wo tra nsport ers lea ds to t he ex c ha nge o f c ytoso lic ADP a nd Pi f or matrix ATP at t he cost o f a n i nflux o f o ne H+

.

The dicar boxylat e carri er ena bles ma lat e, s ucci nat e, a nd fumarat e to be export ed f ro m mitoc ho ndria i n exc ha nge

f or Pi . The tricar boxylat e carri er tra nsports citrat e a nd H+ i n exc ha nge f or ma lat e. Pyr uvat e i n t he c ytoso l ent ers

t he mitoc ho ndria l matrix i n exc ha nge f or OH- (or to get her wit h H+) by mea ns o f t he p yr uvat e carri er . Thes e

mitoc ho ndria l tra nsport ers a nd mor e t ha n f i ve ot hers ha ve a co mmo n str uct ura l moti f. They ar e co nstr uct ed f ro m

t hr ee ta ndem r ep eats o f a 100-r esi due mo dule, eac h co ntai ni ng t wo p utati ve tra ns membra ne s egments

Mitochondrial Transporters. Transporters (also called carriers)are transmembrane proteins that move ions and charged

metabolites across the inner mitochondrial membrane.

Structure of MitochondrialTransporters. Many mitochondrial

transporters consist of three similar

100-residue units. These proteins

contain six putative membrane-

spanning segments.

Mitochondrial carriers



The best current estimates for the

number of protons pumped out of the

matrix by NADH-Q oxidoreductase, Q-cytochrome c oxidoreductase, and

cytochrome c oxidase per electron pair

are four , two, and four , respectively.The synthesis of a molecule of ATP is

driven by the flow of about three protons

through ATP synthase. An additional

proton is consumed in transporting

ATP from the matrix to the cytosol.

Hence, about 2.5 molecules of cytosolic

ATP are generated as a result of the flowof a pair of electrons from NADH to O2.

For electrons that enter at the level of Q-

cytochrome c oxidoreductase, such as those

from the oxidation of succinate or cytosolic

NADH, the yield is about 1.5 molecules of ATP

per electron pair. Hence about 30 molecules of ATP are for med when glucose is com pletely

oxidized to CO2 ; this value supersedes the

traditional estimate of 36 molecules of ATP.



E lectrons do not usually flow through the electron-transport chain to O 2 unless ADP is

simultaneously phosphorylated to ATP. Oxidative phosphorylation requires a supply of NADH (or

other source of electrons at high potential), O2, ADP, and Pi. The most important factor in

determining the rate of oxidative phosphorylation is the level of ADP. The rate of oxygen

consumption by mitochondria increases markedly when ADP is added and then returns to its initial

value when the added ADP has been converted into ATP.

The regulation of the rate of oxidative phosphorylation by the ADP level is called respiratory control

or acceptor control.

T he rate of oxidative phosphorylation is determined by the need of AT P



Electron transport chain and oxidative phosphorylationis inhibited by many chemicals

� Different inhibitors inhibit at different levels.

� Block of ATP synthase«Oligomycin and

Dicyclohexylcarbodimide

� Block of ATP-synthase causes Electron transport

chain to cease.

� Uncouplers e.g. 2,4-dinitrophenol and certain other

acidic aromatic compounds carry

protons across the inner mitochon-

-drial membrane.

� ATP-ADP translocase is specifically inhibited by very

low concentrations of atractyloside (a plant glycoside)

or bongkrekic acid (an antibiotic from a mold).

Atractyloside binds to the translocase when its

nucleotide site faces the cytosol, whereas bongkrekic

acid binds when this site faces the mitochondrial

matrix. Oxidative phosphorylation stops soon after either inhibitor is added, showing that ATP-ADP

translocase is essential.



Regulated Uncoupling Leads to the Generation of HeatBrown fat mitochondria

Action of an Uncoupling Protein. Uncoupling protein-1 (UCP-1) generates heat by

permitting the influx of protons into the mitochondria without the synthesis of ATP.



In addition to UC

P-1, two other uncoupling proteins have been identified. UC

P-2,which is 56% identical in sequence with UCP-1, is found in a wide variety of

tissues. UCP-3 (57% identical with UCP-1 and 73% identical with UCP-2) is

localized to skeletal muscle and brown fat. This family of uncoupling proteins,

especially UC

P-2 and UC

P-3, may play a role in energy homeostasis. In fact, thegenes for UCP-2 and UCP-3 map to regions of the human and mouse

chromosomes that have been linked to obesity, substantiating the notion that

they function as a means of regulating body weight. The use of uncoupling

proteins is not limited to animals, however. The skunk cabbage uses an

analogous mechanism to heat its floral spikes, increasing the evaporation of

odoriferous molecules that attract insects to fertilize its flowers.

Cool facts



Molecular oxygen is an ideal terminal electron acceptor, because its high

affinity for electrons provides a large thermodynamic driving force. However,

danger lurks in the reduction of O 2 . The transfer of four electrons leads to safe

products (two molecules of H2O), but partial reduction generates hazardous

compounds. In particular, the transfer of a single electron to O 2 forms

superoxide anion, whereas the transfer of two electrons yields peroxide.

These compounds and, particularly, their reaction products can be quite harmful to a

variety of cell components. The strategy for the safe reduction of O2 is clear from thediscussion of the reaction cycle: the catalyst does not release partly reduced

intermediates. Cytochrome c oxidase meets this crucial criterion by holding O2 tightlybetween Fe and Cu ions.

Hazards of electron transport chain



T he fix

Superoxide Dismutase Mechanism. The oxidized form

of superoxide dismutase (Mox) reacts with one superoxide

ion to form O2 and generate the reduced form of the

enzyme (Mred). The reduced form then reacts with a

second superoxide and two protons to form hydrogen

peroxide and regenerate the oxidized form of the enzyme.

The hydrogen peroxide formed bysuperoxide dismutase and by other processes is scavenged by catalase, aubiquitous heme protein that catalyzes thedismutation of hydrogen peroxide intowater and molecular oxygen.

Superoxide dismutase and catalase are

remarkably efficient, performing their

reactions at or near the diffusion-limited

rate. Other cellular defenses against

oxidative damage include the antioxidant

vitamins, vitamins E and C. Because it is

lipophilic, vitamin E is especially useful in

protecting membranes from lipid

peroxidation.

Lecture 3 Oxidative Phosphorylation Metabolism III

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