Biological Oxidation by Satish

8/3/2019 Biological Oxidation by Satish

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Life Sciences Study Materials http://biochemden.in Biological Oxidation---------------------------------------------------------------------------------------------------------------------------------

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Prepared by I.Satish Kumar, Lecturer in Biochemistry, http://biochemden.in1

(Life Sciences Study Materials)

(Useful for B.Sc. and M.Sc. Students)

I .Satish K umarEmail: [email protected]

http://biochemden.inFacebook: [email protected]

[email protected]

My Sweet Name: _________________________________________________

Class _______________________ Roll Number ________________________

College Name: ___________________________________________________


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Biological Oxidation

STRUCTURE OF MITOCHONDRIA

HISTORICAL:

1880 - Kolliker - observed in muscle cells of insects

1882 - Flemming- gave the name as fila 1894 - Altamann- gave systematic name observation name as Bioblast 1897-98 Benda- gave name as Mitochondria. He stained the mitochondria with alizarin and crystal violet. 1900 - Michaelis- stained mitochondria with jaunus green 1934 - Bensley & Hoperr said mitochondria is the site for the cellular respiration.

OTHER NAMES:

Fuchsinophilic granules, Parabasal bodies, Plasmosomes, Fila, Chondriosomes, Vernicules Bioblasts.

BIOCHEMISTRY & ANATOMY OF A MITOCHONDRION:

Mitochondria were first observed by Altmann in 1894 who described as bioblasts. Benda(1897) called them mitochondria. (mito

G=thread; chondrion

G=granule). The number of mitochondria

varies with the cell type and functional stages. In eukaryotes, approximately 2000 mitochondriasoccupies one-fifth of its total cell volume. The mitochondrial chemical composition is concerned,mitochondria consist of 65-70% proteins, 25-30% lipids, 5-7% DNA and 0.5%RNA. The4 outermembrane of the mitochondria has porins, which permits molecules upto 10kd. Matrix is gel likesolution, containing high concentration of soluble enzymes, substrate, nucleotide cofactors, ions.

The mitochondrion is a subcellular organelle having the outer and inner membranes enclosingthe matrix. The inner membrane is highly selective in its permeabily characteristics. The innermembrane contains the respiratory chain and translocating systems. The knobs like protrusionsrepresent the ATP synthase system. The inner membrane is folded into a series of internal ridgescalled Cristae, which may be longitudinally or transversely oriented, branched or tabular. Hence, thereare two compartments in mitochondria: the intermembrane space between the outer and innermembranes and the matrix, which is bounded by the inner membrane. Most of the reactions of the TCAcycle and fatty acid oxidation occur in the matrix.

ENZYME LOCALIZATION IN MITOCHONDRIA:

LOCALISATION OF SOME ENZYMES IN RAT-LIVER MITOCHONDRIA:

Outer membrane

Monoamine oxidase

Kynurenine-3-monoxygenase

NADH dehydrogenase

Acyl. CoA Synthetase4 Phospholipase-A2

Nucleoside diphosphate kinase

Space between the membranes: Adenylate kenase Creatine Kinase

Inner membrane:

NADPH dehydrogenase

Iron-Sulfur proteins

Cyt.b,c,c1and aa3 F1 ATPase

Succinate dehydrogenase Carnitine acyl transferase

Matrix:

TCA Cycle enzymes

Fatty acyl-CoA oxidationenzyme.


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Functions of mitochondria:

The mitochondria are organelles which transfer the chemical energy of themetabolites of the cell (through Krebs cycle and the respiratory chain.) into the high-energyphosphate bond of ATP. Thus, mitochondria are the power house of the cell, that producethe4 energy necessary for many vital cellular functions via, motility contraction (musclecontraction), biosynthesis of cell bioluminescence etc.

Mitochondriall Electron transport chain [MtETC]

Prokaryotic cells have no mitochondrial bodies; their plasma membrane appears tobe the site of electron transport and oxidative phosphorylation. Thus, all the cytochromepigment and a number of dehydrogeneous associated with the TCA Cycle, namely Succinic,malic and -KG dehydrogenase, are localized in the bacterial plasma membrane.

In 1948, A.L.Lehninger showed that in the animal cell, the mitochondrion was the solesite for oxidative phosphorylation, the TCA Cycle and fatty acid oxidation.

Components:There are five different kinds of electron carriers that participate in the transport of

electrons from substrates as they are oxidized in the mitochondria. Inb addition, Cu+2

ispresent and functions in the enzyme, Cytochrome oxidase, that catalyzes the reduction of O2

(1) Nicotinamide Nucleotides:

Two pf the oxidations in the TCACycle involve the removal of the equivalent oftwo hydrogen atoms from the substrates,malate and isocitrate. In two others, thosecatalyzed by pyruvate dehydrogenase and -Ketogularate dehydrogenase, the electronsare transferred first to lipoic acid and then via aflavorprotein to NAD

+.

SH2 + NAD+ S + NADH + H

+

(2) Flavoproteins:These proteins contain a very tightly, sometimes covalently bound flavin nucleotide,

either FMN (or) FAD. The oxidized flavin nucleotide can accept either one electron (or) two(yielding FADH2 (or) FMNH2). The standard reduction potential of a flavin nucleotide, unlike

that of NAD (or) NADP, depends on the protein with which it isw associated. The flavinnucleotide should be considered part of the flavoproteins activesite, not as a resultant (or)product6 in the electron-transfer reaction. Because flavoproteins can participate in eitherone-or-two electron transfers, they can serve as intermediate between react6ions in whichtwo electrons are donated and these in which only one electron is accepted.

NADH + H+

+ FMN NAD+

+ FMNH2Succinate + FAD Fumarate + FADH2

(3) Iron Sulfur proteins: This type of protein was first encountered as ferredoxin, a reducing agent involved in

nitrogen fixation and photosynthesis in plants before it was recognized to funct6ion inmt.E.T in animals.

The iron atoms are arranged in paris in an iron-sulfur bridge, which is bounded to the

sulfur atoms of Cysteine residues in the protein. Some iron-Sulfur proteins such as spinach ferredoxins contains only two iron atoms

(Fe2S2) while others contain four (Fe4S4)

(4) Quinones:Mitochondria contain quinine called Ubiquinone (also called Coenzyme.Q

(or) simply Q) which is a lipidsoluble benzoquinone with a long isoprenoid sidechain. Ubiquinone can accept one electron to become the semiquinone radical (QH

*)

or two electrons to form ubiquinol (QH2), it can act at the junction between a twoelectron donor and a one-electron acceptor, because ubiquinone is both small and


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hydrophobic, it is finally diffusible within the lipid bilayer of the inner mitochondrialmembrane and can shuttle reducing equivalents between other, less mobile electroncarriers in the membrane and because it carries both electrons and protons, it plays acentral role coupling in coupling electron flow to proton movement.

(5) Cytochromes:The cytochromes are poteins with characteristic strong absorption of visible

light, due to their iron-containing heme presthatic groups. Mitochondria cantains of

three classes of cytochromes, designated Cyt.a ,Cyt.b and Cyt.c distinguished bydifferences in their light-absorption spectra. Each type of cytochromes in its reduced(Fe

2+) state has three absorption bonds in the visisble range. The longest

wavelength bond is near 600nm in type a.Cyt, near 560nm in type.b and near 550nmin type.c.

The haeme as prostatic group to this cytochromes, but not covalently in Cyt.aand Cyt.b types. In Cyt.c case the haeme group binds tightly by covalently throughCys residues. The Cytochromes of type a and b and some of type C are integralproteins of the inner mitochondrial membrane. Cyt.c of mitochondria, a solubleprotein that associates through electrostatic interactions with the outer surface ofr theinner membrane.

(1) Cytochrome.a and a3: These are also called as Cytochrome oxidase. These are found solely in the

mitochondria. It has molecular weight 72,000 (or) 93,000 Oxidation potential of +0.29Volt. The reduced forms of cytochrome.a of animal tissue exhibit an absorption band

near 600nm. Cytochrome a and a3 possess and identical type of iron-porphyrin complex called

Heme.a,but their location to apo-protein are different. One heme group is located along with one copper ion. This heme is called heme.a.

This cyt.a functions as the anaerobic oxidizing unit. The other heme.a called heme.a3 is located along with the second copper ion at the

binding site for molecular O2 on subunit-I and functions as aerobic reducing unit ofthe enzyme complex.

Cyt-a absorbs at 605,517 and 414nm where as Cyt.a3 absorbs at 600 and 445nm.Cyt .a doest react with O2

Cyt.oxidase thus constitute the last carrier in the chain of electron transport and isreferred to as the terminal oxidase4 of the cytochrome chain.

(2) Cytochrome-b: Cyt.b contains protoporphyrin IX complex, but the apoprotein is different. Cyt.b of animal tissue has -absorption bands near 563nm, -absorption bands near

530nm and -absorption near 430nm. It is thermostable and not easily extractable Oxidation potential is +0.04 volot in the mitochondria, and -0.34 volt when it is free. Cyt.b2,b3,b4 etc are found in microorganisms Thje Cyt.b doest react with O2, CO (or) CN

-

Cyt+.b is reduced by accepting an electron from reduced COQ.

(3)Cytochrome.C: It is the best characterized of all cytochromnes. It is water soluble and easily extractable. The Cyt.C have - absorption bands near 550nm, -absorption bands near 521nm

and -absorption near 416nm In Cyt.C heme is attached with protein by means of two thioesther linkages involving

sulphur of two cysteine and apoprotein. It is a basic protein with one polypeptide chain with 104a.as.


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Cyt.C acts as an electron carrier because its iron atom readily changes its valancefrom 3to 3, i.e., Fe

+3+e

-Fe

+2.

THE RESPIRATORY CHAINThe electron carriers of the respiratory chain are organized into the membrane

embedded supromolecular complexes that can be physically separated. Gentle treatment ofthe inner mitochondrial membrane with detergents allows the resolution of four unique

electron carrier complexes, each capable of catalyzing electron transfer through a portion ofthe chain.

Complex I and II catalyze electron transfer to ubiquinone from two different electrondonors: NADH (complex .I) and succinate (Complex .II) Complex .III carriers electrons fromubiquinone to cytochrome.c, and complex. IV completes the sequence by transferreingelectrons from Cyt.C to O2

PROTEIN COMPONENTS OF THE MITOCHONDRIAL ETC:

Enzyme Complex Mass(KD)

Numberof

subunits

Prostheticgroup(s)

Complex-I: NADH dehydrogenaseComplex-II:Succinate dehydrogenase

Complex-III: Ubiquinone: Cytochrome.Coxidoreductase, Cytochrome.C

Complex-IV:Cytochrome oxidase

850140

250

13160

42(14)5

11

113(3-4)

FMN, Fe-SFAD,Fe-S

Hemes,Fe-S

HemeHemes;CUA,CUB

Complex I:Complex-I also called NADH: Ubiquinine oxidoreductase is a large enzyme

composed of 42 different polypeptide chains, including as FMN-containing flavoprotein and atleast six iron-sulfur centers. The complex shows L-shaped, arm extending into the matrix.

Mechanism:Complex-I catalyzes the transfer of a hydride ion from NADH to FMN, from which two

electrons pass through a series of Fe-5 centers to the iron-sulfur protein N-2 in the matrixarm of the complex. Electron transfer from N-2 to ubiquinone on the membrane arm formsQH2, which diffuses into the lipid bilayer. It alos drives the expulsion from the matrix of fourprotons per papir of electrons. The detailed mechanism that couples electron and protontransfer in complex-I is not yet known,but probably involves a Q cycle similar to that incomplex-III in which QH2 participates twice per electron pair. This proton flux produces anelectrochemical potential across theinner mitochondrial membrane (N-sidenegative, P-side positive), whichconserves some of the energy releasedby the electron transfer reactions. Thiselectrochemical pote4ntial drives ATPsynthesis. There is a large negative freeenergy change, the energy released is -

12K.Cal/mol. Utilized by ADP&P formsATP.

NADH FMN (Fe-S1) (Fe-S2) (Fe-S3) (Fe-S4)CoQ


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Complex-II: Succinate dehydrogenase:Complex-II catalyzes the reduction of Co.Q by electrons remove from succinate.

Succinate + CoQ Fumarate + CoQ.H2

This complex which contains FAD, is composed of four polypeptides with molecularweight of 70,000, 27,000, 15,000 and 13,000. Succinate dehydrogenase, the onlymembrane-bound enzyme in the citric acid cycle. Although smaller and simpler than

complex-I, It contains two types of prosthetic groups and at least four different proteins. Oneprotein has a covalently bound FAD and an Fe-S center with four Fe atoms; a second iron-sulfur protein is also present. Electrons pass from succinate to FAD, then through the Fe-Scenters to ubiquinone. Other substrates for mitochondrial dehydrogenases pass electronsinto the respiratory chain at the level of ubiquinone, but not through complex-II. The enzymesare acyl.CoA dehydrogenase and Glycerol-3-Pdehydrogenase4. The acyl.CoAdehydrogenase involving electron transfer proteins are ETF (electron transferringflavoprotein): Ubiquinone oxidoreductase. QH2 from all these reactions is reoxidised bycomplex-III, the next component in the mitochondrial electron-transfer chain.

Complex-III: Ubiquinone: Cytochrome.C.Oxidoreductase:The Complex-III couples thetransfer of electrons from ubiquinol(QH2) to

cytochrome.C with the vectorial transport of protons from the matrix to the intermembranespace. This is a multi-protein complex, consisting of a cluster of iron-sulfur proteins, Cyt.band Cyt.C1. Cyt.b & C1 contain heme prosthetic group. During this process of transfer ofelectron, the iron in heme group shuttles between Fe

+3and Fe

+2forms. The free energy

change is -10Kcal/mol; one molecule of ATP is synthesized in this step.

Cytochrome.C:It contains one heme prosthetic group. The term cytochrome is derived from a greek

word meaning Cellular colors. Axel Theorell isolated it. It is not a part of an enzymecomplex, it moves between complex.III and IV as a freely soluble protein. Cyt.C collects

electrons from complex.III and delivers them to complex.IV. Cyt.C also the mediator ofapoptosis (Programmed cell death).

QH2 + Cyt.c Q + Cyt.C(red) (oxi) (oxi) (red)

Complex-IV: Cytochrome Oxidase:In the final step of the respiratory chain, complex IV carries electrons from

cytochrome.C to molecular oxygen, reducing it to H2O. The complex IV is tightly bound to themitochondrial membrane. Four electrons are accepted from Cytochrome.C, and passed on to


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molecular oxygen. Complex.IV also functions as a proton pump; free energy change is -24Kcal/mol and 1ATP molecule is synthesized.

Cyt.oxidase contains two heme groups and two copper ions. The3 two heme groupsare structurally similar, but they are located at different parts of the enzyme complex anddenoted as Cyt.a and Cyt.a3. The functional unit of the enzyme is a single protein and isreferred to as Cytochrome-a,a3.

Path of electron through Complex-IV:

The three proteins critical to electron flow are I, II and III. The lighter outline includesthe other ten proteins in the complex. Electron transfer through complex-IV begins when twomolecules of reduced Cyt.C each donates an electron to the binuclear centre CuA. Fromhere electrons pass through heme.a to the Fe-Cu center (Cyt.a3 & CuB). Oxygen now bindsto heme a3 and I reduced to its peroxy derivative (O2

2-) by two electrons from the Fe-Cu

center. Delivery of two more electrons from Cyt.C converts the (O22-

) to two molecules ofwater, consuming four Substrate protons from the matrix. At the same time, four moreprotons are pumped from the matrix by an as yet unknown mechanism.


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Inhibitors of ETC:The inhibitors that arrest respiration by combining with members of the respiratory

chain, rather than with the enzymes that may be involved in coupling respiration with ATPsynthesis. The various inhibitors of this category:

NADH

Site I [FMN] Rotenone Piencidin.A[Fe-S] Amytal

Q

Site II [Cyt.b] Antimycin.A Dimercaprol[Cyt.C1]

Cyt.C

CN-

Site III N3- H2S

Oxygen CO

Inhibitors of electron transport chain:Transfer of electrons is selectively inhibited as various components of the electron

transport chain by a variety of substances. Some of these are used as poisons (eg:insecticides) and some of which are used as drugs.

Site-I (Complex-I):

Rotenone: A fish poison and also insecticide. Inhibits transfer of electrons throughComplex-I-NADH-Q-reductase.

Amobarbital (Amytal) and Secobarbital: Inhibits electrons transfer by competingwith Co.Q.

Piericidin.A: An antibiotic, Blocks electron transfer by competing with Co.Q. Drugs: Chlorpromazine and hypotensive drug like guanethidine.

Step II (Complex-III): Antimycin.A BAL (dimo-Caprol) Hypoglycamic drugs: like phenformin

Step III (Complex-IV):

Cyanide(CN-)

H2S Azide (N3

-)

CO (Carbon monoxide): It inhibits Cyt .oxidase by combining with O2 binding site.It can be reversed by illumination with light.

Complex-II (Succinate dehydrogenase: ( FAD):

Carboxin TTFA Malonate: A competitive inhibitor of succinate dehydrogenase


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OXIDATIVE PHOSPHORYLATION

The coupling of oxidation with phosphorylation is termed Oxidative phosphorylation, which takesplace during the electron transport in the inner membrane.

DEFINITION:The endergonic synthesis of ATP from ADP and p

iin mitochondria is called oxidative

phosporylation, which is catalyzed by the enzyme ATP synthase (or) mitochondrial ATPase (or) H+-

ATPase because it was discovered through its catalysis of hydrolytic reaction.

HYPOTHESIS:

Many hypothesis have been formulates to explain the coupling of electron-transpoort and ATPsynthesis. But some are discussed below

(1) Chemical coupling hypothesis:

It was proposed by Edward Slater & Lehninger (1967). According to this synthesiselectron transport yields reactive intermediates whose break down drives oxidativephosphorylation.

Eg:In glycolysis, oxidation of Gly-3-P by NAD+

gives 1,3-bis P glycerate. Its phosphate groupis transferred from Enzyme intermediate to ADP.

The difficulty with this mechanism is no appropriate reactive intermediate have been identified.So, the hypothesis not agreed by so many scientists.

(2) Conformational coupling hypothesis:

It was formed by Poul boyer. According to this hypothesis, electron transport cause4sactivation of proteins of inner mitochondrial membrane. These activated proteins in some wayassociated with ATP synthase. The retention of activate4d protein, drives the ATP synthesis.The disadvantage with this mechanism is, it has little experimental support.

(3) Chemiosmotic Hypothesis:

Peter Mitchell in 1961 (Nobel prize, 1978) proposed the chemiosmotic theory to explain theoxidative phosphorylation. The transport of electrons from inside to outside of inner-mitochondrial membrane is accompanied by the generation of a proton gradient across themembrane. Protons accumulate outside the membrane, creating electrochemical potentialdifference. This proton motive force (pmf) drives the synthesis of ATP by ATP synthasecomplex.

Mitchells hypothesis, oxidative phosphorylations are coupled by a part on gradient is nowsupported by a wealth of evidence:

(a) Electron transport generates a proton gradient across the inner mitochondrialmembrane. The P

Houtside is 1.4units lower than inside and the membrane

potential is 0.14V, the outside being positive.(b) ATP is synthesized when a P

Hgradient is impose4d on mitochondria in the

absence of electron transport.(c) NADH.Q reductase, Cyt. Reductase and Cyt.oxidse pump protons out of the

matrix. Their return drives ATP formation by ATP synthase.(d) A closed compartment is essent ial for oxidative phosphorylation. ATP synthesis

coupled to electron transfer doesnt occur in soluble preparations or in membrane4fragments lacking well-defined inside and outside compartments.

Two theories established to explain the pumping of protons.

(i) Redox loop mechanism(ii) Proton transport mechanism

(i)Redox loop mechanism:

Mitchell has proposed a fantastic scheme which is based on the first that reducing onthe fact that reducing equivalents are transferred as H atoms by some of the electron carriers(such as Fe-S centre and cytochromes). He opined that hydrogen carrying and electroncarrying proteins alternate in the respiratory chain to form 3 functional loops called theoxidation-reduction loops (=O/R loops). Each loop corresponds functionally to the couplingsites I, II and III of the chemical hypothesis respective4ly. In each loop, 2 protons are carried out


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through the loop from Matrix to intermembrane space; the corresponding pair of electrons is thencarried back from the outer to the inner surface of the membrane. Each pair of reducingequivalents provides the osmotic energy to make one mole of ATP.

(ii) Proton transfer mechanism:

In the electron transport, as envisaged by Peter Mitchell, the respiratory chain is folded into 3oxidation-reduction (o/r) loops. It is assumed that the members of the respiratory chain areorganized in the membrane to provide the necessary sidedness. Each pair of electronstransferred from NADH to oxygen causes 6 protons to be translocated from inside to the outside

of the coupling membrane.

INHIBITORS AND COUPLORS OF OXIDATIVE PHOPHORYLATION:

The following compounds inhibit both electron transport and oxidative phosphorylation.

Oligomycins: Is a polypeptide antibiotic are obtained from various species of Streptomyces.They inhibit the transfer of high-energy phosphate to ADP and also inhibit e4lectron

Transfers coupled to phosphorylation.The antibiotic is potent inhibitor to ATP synthase complex.

Rutamycin:

This antibiotic also inhibits both ETC and oxidative phosphorylation. Attractylate:

It backs oxidativephosphorylation by compeling with ATP & ADP for a site on the ADP-ATPantiport of the mitochondrial membranes.

Bongkrekate:

It is a toxin formed by bacteria (Pseudomonas) in a coconut preparation from Java.1 It also blocks the ADP-ATP antiport.

2,4-Dinitrophenol:

A classic uncoupler of oxidative phosphorylation. The substance carries protons across the inner mitochondria membrane. In the presence of these uncouplers, electron transport from NADH to O2 proceeds normally,but ATP is not formede by the mitochondria. ATP are because the proton motive force acrossthe inner mitochondrial membrane is dissipated. DNA and other uncoupllers are very useful in metabolic studies because of their specificeffect on outside phosphorylation.

Dicoumarol (Vitamin.K analogue):Used as anticoagulant.

Calcium:Transport of Ca+2 ion into mitochondria can cause uncoupling.

1. Mitochondrial transport of Ca+2

is energetically coupled to oxidative phosphorylation.2. It is coupled with uptake of p

i

3. When calcium is transported into mitochondria, electron transport can proceed but energy isrequired to pump the4 Ca

+2into the mitochondria. Hence, no energy is stored as ATP.

CCCP (Chloro carbonyl cyanide phenyl hydrazone):

Most active uncouplerThese lipid soluble substances can carry protons across the inner mitochondrial membrane.

Focus:The uncoupling of oxidative phosphorylation can be biologically useful. It is a

means of generating heat to maintain body temperature in hibernating animals, some newborn

animals (including humans) and mammals added to cold. Brown adipose tissue, which is veryrich in mitochondria, is specialized for this process of Thermo genesis. The inner

mitochondria membrane of these mitochondria contains a large amount of Thermogenin(also called the uncoupling protein), a dimmer of 33-kd submits that resembles the ATP-ADP translocase. Thermogenin forms a path way for the few of protons from the cytosol to

the matrix. In essence, thermogenin generates heat by short-circuiting the mitochondrialproton battery. This dissipative proton pathway is activated by free fatty acids liberated from

triacylglycerides in response to hormonal signals {When an uncoupler is added, there is

marked increase in O2 uptake].


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Valinomycin:

Produced by a type of streptomycesTransports K

+from the cytosol into matrix and H

+from matrix to cytosol, thereby decreasing

the proton gradient.Physiological un-couplers:

(a) Excessive thyroxin hormone(b) EFA deficiency(c) Long chain FA in brown adipose tissue

(d) Unconjugated hyperbilirubinaemia

CHEMIOSMOTIC THEORY OF ATP SYNTHESIS:

ATP is synthesized by an enzyme complexmade of a proton-conducting F0 unit and a catalystF1 unit. The mitochondrial inner membranecontains the ATP synthesizing enzyme complexcalled ATP synthase(or) F0 F1-ATPase. (F forfactor). F1 component is like a doorknobprotruding into the matrix from the inner membrane.It is attached by a stalk to F0 component, which isembedded in the inner membrane and extendsacross it, (O denotes, it is the protein of enzymewhich binds the toxic antibiotic Oligomycin). Thusthe physiological role of the F1 component is to

catalyze the synthesis of ATP.

The spheric F1 component (MW=360Kdal) contains a polypeptide chain subunits of five kinds(designated as ,,, and ) arranged into a cluster. It has many binding site4s for ATP and ADP. Thecuboidal F0component is a hydrophobic segment of 4polypeptide chains. F0 is the proton channel of theenzyme complex. The stalk is the communicating portion of the enzyme complex.

Binding change mechanism:

The mechanism of ATP synthesis byproton-translocating ATP synthase can beconceptually broken down into three phases:(1) Translocation of protons carried out by F0(2) Catalysis of formation of the phosphor-

anhydride bond of ATP carried out by F1.(3) Coupling of the dissipation of the proton

gradient with ATP synthesis, which requires

interaction of F1 and F0.

The available evidence supports amechanism for ATP formation proposed by PaulBoyer. According to this binding changemechanism, F1 has three interacting catalyticpromoters, each in a different conformational state:

L-State: That binds substrates and products loosely.T-State: That binds them tightly.O-State: That does not bind them at all (open state).

The free energy released on protontranslocation is harnessed to interconvert these4 threestates. The phosphor anhydride bond of ATP issynthesized only in the T-State, and ATP is releasedonly in the O-State. The reaction involves three steps.

(1) ADP and pibond to the loose (L) binding site.

(2) A free energy driven conformational changeconverts the L-site to a tight (T) binding site thatcatalyzes the format6ion of ATP. This step alsoinvolves conformational changes of the other twopromoters that convert the ATP-containing T-site toan open (o) site and convert the O site to an L-site.


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(3) ATP is synthesized at the T-site on one subunit while ATP dissociates from the3 O-site on anothersubunit. The free energy supplied by the proton flow primarily facilitates the release of the newlysynthesized ATP from the enzyme; that is, it drives the T O transition thereby disrupting the enzyme-ATP interactions that had been promoted the spontaneous formation of ATP from ADP and p

iin the T-

site.

Reference Books:

1. Biochemistry, Voet and Voet, 2/e

2. Principles of Biochemistry, Lehninger, Nelson & Cox, 3/e (Worth)3. Biochemistry, Stryr, 4/e, Whfreeman Publications.4. Cell biology, Shrma, SChands Publications.

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Biological Oxidation by Satish