Life Sciences Study Materials Biological Oxidation


(Life Sciences Study Materials)
(Useful for B.Sc. and M.Sc. Students)

I.Satish Kumar

My Sweet Name: _________________________________________________ Class _______________________ Roll Number ________________________ College Name: ___________________________________________________ ------------------------------------------------------------------------------------------------------Prepared by I.Satish Kumar, Lecturer in Biochemistry, 1

Life Sciences Study Materials Biological Oxidation ---------------------------------------------------------------------------------------------------------------------------------

Biological Oxidation
STRUCTURE OF MITOCHONDRIA HISTORICAL:        1880 1882 1894 1897-98 1900 1934 - Kolliker - observed in muscle cells of insects - Flemming- gave the name as “fila” - Altamann- gave systematic name observation name as “Bioblast” – Benda- gave name as “Mitochondria”. He stained the mitochondria with “alizarin” and “crystal violet”. - Michaelis- stained mitochondria with “jaunus green” - Bensley & Hoperr – said mitochondria is the site for the cellular respiration.

OTHER NAMES:        Fuchsinophilic granules, Parabasal bodies, Plasmosomes, Fila, Chondriosomes, Vernicules Bioblasts.

Mitochondria were first observed by “Altmann” in 1894 who described as “bioblasts”. Benda G G (1897) called them “mitochondria”. (mito =thread; chondrion =granule). The number of mitochondria varies with the cell type and functional stages. In eukaryotes, approximately 2000 mitochondrias occupies 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 outer membrane of the mitochondria has “porins”, which permits molecules upto 10kd. Matrix is gel like solution, containing high concentration of soluble enzymes, substrate, nucleotide cofactors, ions. The mitochondrion is a subcellular organelle having the outer and inner membranes enclosing the matrix. The inner membrane is highly selective in its permeabily characteristics. The inner membrane contains the respiratory chain and translocating systems. The knobs like protrusions represent the ATP synthase system. The inner membrane is folded into a series of internal ridges called “Cristae”, which may be longitudinally or transversely oriented, branched or tabular. Hence, there are two compartments in mitochondria: the intermembrane space between the outer and inner membranes and the matrix, which is bounded by the inner membrane. Most of the reactions of the TCA cycle and fatty acid oxidation occur in the matrix.

Outer membrane  Monoamine oxidase  Kynurenine-3-m onoxygenase  NADH dehydrogenase  Acyl. CoA Synthetase4  Phospholipase-A2  Nucleoside diphosphate kinase Inner membrane:      

Space between the membranes:  Adenylate kenase  Creatine Kinase Matrix:

NADPH dehydrogenase Iron-Sulfur proteins Cyt.b,c,c1and aa3 F1 ATPase Succinate dehydrogenase Carnitine acyl transferase

 

TCA Cycle enzymes Fatty acyl-CoA oxidation enzyme.

------------------------------------------------------------------------------------------------------Prepared by I.Satish Kumar, Lecturer in Biochemistry,


Life Sciences Study Materials Biological Oxidation --------------------------------------------------------------------------------------------------------------------------------Functions of mitochondria: The mitochondria are organelles which transfer the chemical energy of the metabolites of the cell (through Krebs cycle and the respiratory chain.) into the high-energy phosphate bond of ATP. Thus, mitochondria are the “power house of the cell”, that produce the4 energy necessary for many vital cellular functions via, motility contraction (muscle contraction), biosynthesis of cell bioluminescence etc. Mitochondriall Electron transport chain [MtETC] Prokaryotic cells have no mitochondrial bodies; their plasma membrane appears to be the site of electron transport and oxidative phosphorylation. Thus, all the cytochrome pigment and a number of dehydrogeneous associated with the TCA Cycle, namely Succinic, malic and α-KG dehydrogenase, are localized in the bacterial plasma membrane. In 1948, A.L.Lehninger showed that in the animal cell, the mitochondrion was the sole site 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 +2 electrons from substrates as they are oxidized in the mitochondria. Inb addition, Cu is present and functions in the enzyme, Cytochrome oxidase, that catalyzes the reduction of O2 (1) Nicotinamide Nucleotides: Two pf the oxidations in the TCA Cycle involve the removal of the equivalent of two hydrogen atoms from the substrates, malate and isocitrate. In two others, those catalyzed by pyruvate dehydrogenase and αKetogularate dehydrogenase, the electrons are transferred first to lipoic acid and then via a flavorprotein 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 flavin nucleotide should be considered part of the flavoproteins activesite, not as a resultant (or) product6 in the electron-transfer reaction. Because flavoproteins can participate in either one-or-two electron transfers, they can serve as intermediate between react6ions in which two electrons are donated and these in which only one electron is accepted. NADH + H + FMN Succinate + FAD

 

NAD + FMNH2 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 in mt.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 side * chain. 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 two electron donor and a one-electron acceptor, because ubiquinone is both small and

------------------------------------------------------------------------------------------------------Prepared by I.Satish Kumar, Lecturer in Biochemistry,


Life Sciences Study Materials Biological Oxidation --------------------------------------------------------------------------------------------------------------------------------hydrophobic, it is finally diffusible within the lipid bilayer of the inner mitochondrial membrane and can shuttle reducing equivalents between other, less mobile electron carriers in the membrane and because it carries both electrons and protons, it plays a central 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 by differences in their light-absorption spectra. Each type of cytochromes in its reduced (Fe2+) 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 550nm in type.c. The haeme as prostatic group to this cytochromes, but not covalently in Cyt.a and Cyt.b types. In Cyt.c case the haeme group binds tightly by covalently through “Cys” residues. The Cytochromes of type a and b and some of type “C” are integral proteins of the inner mitochondrial membrane. Cyt.c of mitochondria, a soluble protein that associates through electrostatic interactions with the outer surface ofr the inner 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 α 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 of the enzyme complex.  Cyt-a absorbs at 605,517 and 414nm where as Cyt.a3 absorbs at 600 and 445nm. Cyt .a does’t react with O2  Cyt.oxidase thus constitute the last carrier in the chain of electron transport and is referred 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 α-absorption bands near 563nm, β-absorption bands near 530nm and γ-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 does’t 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 α- absorption bands near 550nm, β-absorption bands near 521nm and γ-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

------------------------------------------------------------------------------------------------------Prepared by I.Satish Kumar, Lecturer in Biochemistry,


Life Sciences Study Materials Biological Oxidation -------------------------------------------------------------------------------------------------------------------------------- Cyt.C acts as an electron carrier because its iron atom readily changes its valance +3 +2. from 3to 3, i.e., Fe +e Fe

The electron carriers of the respiratory chain are organized into the membrane embedded supromolecular complexes that can be physically separated. Gentle treatment of the inner mitochondrial membrane with detergents allows the resolution of four unique electron –carrier complexes, each capable of catalyzing electron transfer through a portion of the chain. Complex I and II catalyze electron transfer to ubiquinone from two different electron donors: NADH (complex .I) and succinate (Complex .II) Complex .III carriers electrons from ubiquinone to cytochrome.c, and complex. IV completes the sequence by transferreing electrons from Cyt.C to O2 PROTEIN COMPONENTS OF THE MITOCHONDRIAL ETC: Enzyme Complex Mass (KD) 850 140 250 Number of subunits 42(14) 5 11 Prosthetic group(s) FMN, Fe-S FAD,Fe-S Hemes,Fe-S

Complex-I: NADH dehydrogenase Complex-II:Succinate dehydrogenase Complex-III: Ubiquinone: Cytochrome.C oxidoreductase, Cytochrome.C Complex-IV:Cytochrome oxidase

13 160

1 13(3-4)

Heme Hemes;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 at least 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 matrix arm of the complex. Electron transfer from N-2 to ubiquinone on the membrane arm forms QH2, which diffuses into the lipid bilayer. It alos drives the expulsion from the matrix of four protons per papir of electrons. The detailed mechanism that couples electron and proton transfer in complex-I is not yet known,but probably involves a Q cycle similar to that in complex-III in which QH2 participates twice per electron pair. This proton flux produces an electrochemical potential across the inner mitochondrial membrane (N-side negative, P-side positive), which conserves some of the energy released by the electron transfer reactions. This electrochemical pote4ntial drives ATP synthesis. There is a large negative free energy change, the energy released is 12K.Cal/mol. Utilized by ADP&P forms ATP. NADH  FMN  (FeS1) (Fe-S2) (Fe-S3) (FeS4)CoQ

------------------------------------------------------------------------------------------------------Prepared by I.Satish Kumar, Lecturer in Biochemistry,


Life Sciences Study Materials Biological Oxidation --------------------------------------------------------------------------------------------------------------------------------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 molecular weight of 70,000, 27,000, 15,000 and 13,000. Succinate dehydrogenase, the only membrane-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. One protein has a covalently bound FAD and an Fe-S center with four Fe atoms; a second ironsulfur protein is also present. Electrons pass from succinate to FAD, then through the Fe-S centers to ubiquinone. Other substrates for mitochondrial dehydrogenases pass electrons into the respiratory chain at the level of ubiquinone, but not through complex-II. The enzymes are “acyl.CoA dehydrogenase” and “Glycerol-3-Pdehydrogenase4”. The acyl.CoA dehydrogenase involving electron transfer proteins are “ETF (electron transferring flavoprotein): Ubiquinone oxidoreductase”. QH2 from all these reactions is reoxidised by complex-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 intermembrane space. This is a multi-protein complex, consisting of a cluster of iron-sulfur proteins, “Cyt.b” and “Cyt.C1”. Cyt.b & C1 contain heme prosthetic group. During this process of transfer of electron, the iron in heme group shuttles between Fe+3 and Fe+2 forms. 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 enzyme complex, 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 of ‘apoptosis’ (Programmed cell death).

QH2 + Cyt.c 
(red) (oxi)

Q + Cyt.C
(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 the mitochondrial membrane. Four electrons are accepted from Cytochrome.C, and passed on to

------------------------------------------------------------------------------------------------------Prepared by I.Satish Kumar, Lecturer in Biochemistry,


Life Sciences Study Materials Biological Oxidation --------------------------------------------------------------------------------------------------------------------------------molecular oxygen. Complex.IV also functions as a proton pump; free energy change is -24 Kcal/mol and 1ATP molecule is synthesized. Cyt.oxidase contains two heme groups and two copper ions. The3 two heme groups are structurally similar, but they are located at different parts of the enzyme complex and denoted as “Cyt.a” and “Cyt.a3”. The functional unit of the enzyme is a single protein and is referred 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 includes the other ten proteins in the complex. Electron transfer through complex-IV begins when two molecules of reduced Cyt.C each donates an electron to the binuclear centre ‘CuA’. From here electrons pass through heme.a to the Fe-Cu center (Cyt.a3 & CuB). Oxygen now binds 2to heme a3 and I reduced to its peroxy derivative (O2 ) by two electrons from the Fe-Cu 2center. Delivery of two more electrons from Cyt.C converts the (O2 ) to two molecules of water, consuming four “Substrate” protons from the matrix. At the same time, four more protons are pumped from the matrix by an as yet unknown mechanism.

------------------------------------------------------------------------------------------------------Prepared by I.Satish Kumar, Lecturer in Biochemistry,


Life Sciences Study Materials Biological Oxidation ---------------------------------------------------------------------------------------------------------------------------------

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 ATP synthesis. The various inhibitors of this category: NADH ↓ Site I [FMN] ────── [Fe-S] ↓ Q ↓ Site II [Cyt.b] ────── [Cyt.C1] ↓ Cyt.C ↓ Site III ────── ↓ Oxygen

Rotenone Piencidin.A Amytal

Antimycin.A Dimercaprol



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 through “Complex-I-NADH-Q-reductase”.  Amobarbital (Amytal) and Secobarbital: Inhibits electrons transfer by competing with 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

------------------------------------------------------------------------------------------------------Prepared by I.Satish Kumar, Lecturer in Biochemistry,


Life Sciences Study Materials Biological Oxidation ---------------------------------------------------------------------------------------------------------------------------------

The coupling of oxidation with phosphorylation is termed “Oxidative phosphorylation”, which takes place during the electron transport in the inner membrane. DEFINITION: The endergonic synthesis of ATP from ADP and p in 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 ATP synthesis. But some are discussed below (1) Chemical coupling hypothesis: It was proposed by “Edward Slater” & “Lehninger (1967)”. According to this synthesis electron transport yields reactive intermediates whose break down drives oxidative phosphorylation. + Eg:In glycolysis, oxidation of Gly-3-P by NAD gives 1,3-bis P glycerate. Its phosphate group is 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 cause4s activation of proteins of inner mitochondrial membrane. These activated proteins in some way associated 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 the oxidative phosphorylation. “The transport of electrons from inside to outside of innermitochondrial membrane is accompanied by the generation of a proton gradient across the membrane. Protons accumulate outside the membrane, creating “electrochemical potential difference”. This proton motive force (pmf) drives the synthesis of ATP by ATP synthase complex. Mitchell’s hypothesis, oxidative phosphorylations are coupled by a part on gradient is now supported by a wealth of evidence: (a) Electron transport generates a proton gradient across the inner mitochondrial H membrane. The P outside is 1.4units lower than inside and the membrane potential is 0.14V, the outside being positive. H (b) ATP is synthesized when a P gradient 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 essential for oxidative phosphorylation. ATP synthesis coupled to electron transfer doesn’t occur in soluble preparations or in membrane4 fragments lacking well-defined inside and outside compartments. Two theories established to explain the pumping of protons. (i) (ii) Redox loop mechanism Proton transport mechanism

(i)Redox loop mechanism: ‘Mitchell’ has proposed a fantastic scheme which is based on the first that reducing on the 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 electron carrying proteins alternate in the respiratory chain to form 3 “functional loops” called the “oxidation-reduction loops” (=O/R loops). Each loop corresponds functionally to the coupling sites I, II and III of the chemical hypothesis respective4ly. In each loop, 2 protons are carried out

------------------------------------------------------------------------------------------------------Prepared by I.Satish Kumar, Lecturer in Biochemistry,


Life Sciences Study Materials Biological Oxidation --------------------------------------------------------------------------------------------------------------------------------through the loop from Matrix to intermembrane space; the corresponding pair of electrons is then carried back from the outer to the inner surface of the membrane. Each pair of reducing equivalents 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 3 oxidation-reduction (o/r) loops. It is assumed that the members of the respiratory chain are organized in the membrane to provide the necessary sidedness. Each pair of electrons transferred from NADH to oxygen causes 6 protons to be translocated from inside to the outside of the coupling membrane.

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-ATP antiport 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 O 2 proceeds normally, but ATP is not formede by the mitochondria. ATP are because the proton motive force across the inner mitochondrial membrane is dissipated.  DNA and other uncoupllers are very useful in metabolic studies because of their specific effect on outside phosphorylation.

 

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 very rich 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 “ATPADP 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 mitochondrial proton 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].   Dicoumarol (Vitamin.K analogue): Used as anticoagulant. Calcium:Transport of Ca+2 ion into mitochondria can cause uncoupling. +2 1. Mitochondrial transport of Ca is energetically coupled to oxidative phosphorylation. i 2. It is coupled with uptake of p 3. When calcium is transported into mitochondria, electron transport can proceed but energy is +2 required to pump the4 Ca into the mitochondria. Hence, no energy is stored as ATP. CCCP (Chloro carbonyl cyanide phenyl hydrazone): Most active uncoupler These lipid soluble substances can carry protons across the inner mitochondrial membrane.

------------------------------------------------------------------------------------------------------Prepared by I.Satish Kumar, Lecturer in Biochemistry,


Life Sciences Study Materials Biological Oxidation -------------------------------------------------------------------------------------------------------------------------------- Valinomycin: Produced by a type of streptomyces + + Transports 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

ATP is synthesized by an enzyme complex made of a proton-conducting F 0 unit and a catalyst F1 unit. The mitochondrial inner membrane contains the ATP synthesizing enzyme complex called ‘ATP synthase’(or) ‘F0 F1-ATPase’. (F for factor). F1 component is like a “doorknob” protruding into the matrix from the inner membrane. It is attached by a stalk to F0 component, which is embedded in the inner membrane and extends across it, (“O” denotes, it is the protein of enzyme which binds the toxic antibiotic “Oligomycin”). Thus the 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 α,β,γ,δ and ε) arranged into a cluster. It has many binding site4s for ATP and ADP. The cuboidal F0component is a hydrophobic segment of 4polypeptide chains. F0 is the proton channel of the enzyme complex. The stalk is the communicating portion of the enzyme complex. Binding change mechanism: The mechanism of ATP synthesis by proton-translocating ATP synthase can be conceptually broken down into three phases: (1) Translocation of protons carried out by “F0” (2) Catalysis of formation of the phosphoranhydride 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 a mechanism for ATP formation proposed by “Paul Boyer”. According to this “binding change mechanism”, F1 has three interacting catalytic promoters, 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 proton translocation is harnessed to interconvert these4 three states. The phosphor anhydride bond of ATP is synthesized only in the “T-State”, and ATP is released only in the “O-State”. The reaction involves three steps. (1) ADP and p bond to the loose (L) binding site. (2) A free energy driven conformational change converts the L-site to a tight (T) binding site that catalyzes the format6ion of ATP. This step also involves conformational changes of the other two promoters that convert the ATP-containing T-site to an open (o) site and convert the “O” site to an “L”site.

------------------------------------------------------------------------------------------------------Prepared by I.Satish Kumar, Lecturer in Biochemistry,


Life Sciences Study Materials Biological Oxidation --------------------------------------------------------------------------------------------------------------------------------(3) ATP is synthesized at the T-site on one subunit while ATP dissociates from the3 O-site on another
subunit. The free energy supplied by the proton flow primarily facilitates the release of the newly synthesized ATP from the enzyme; that is, it drives the T O transition thereby disrupting the ‘enzymei ATP’ interactions that had been promoted the spontaneous formation of ATP from ADP and p in the Tsite.

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, SChand’s Publications.

------------------------------------------------------------------------------------------------------Prepared by I.Satish Kumar, Lecturer in Biochemistry,