Principles of BIOCHEMISTRY - biochem.vsmu.edu.uabiochem.vsmu.edu.ua/2_med_biochem_e/lecture_biological-oxidation.pdf · 3.Interaction between organs. 4.Cell (membrane) ... cycle to

BioenergeticsBioenergetics

The tiny hummingbirds can store enough fuel to fly a distance of 500 miles without resting. This achievement is possible because of the ability to convert fuels into the cellular energy currency, ATP.

• Metabolism - the entire network of chemical reactions carried out by living cells. Metabolism also includes coordination, regulation and energy requirement.

• Metabolites - small molecule intermediates in the degradation and synthesis of polymers Most organism use the same general pathway for extraction and utilization of energy.

All living organisms are divided into two major classes:

Autotrophs – can use atmospheric carbon dioxide as a sole source of carbon for the synthesis of macromolecules. Autotrophs use the sun energy for biosynthetic purposes. Heterotrophs – obtain energy by ingesting complex carbon-containing compounds.

Heterotrophs are divided into aerobs and anaerobs.

(a) Linear (b) Cyclic

(c) Spiral pathway (fatty acid biosynthesis)

A sequence of reactions that has a specific purpose (for instance: degradation of glucose, synthesis of fatty acids) is called metabolic pathway.

Metabolic pathway may be:

Catabolic reactions - degrade molecules to create smaller molecules and energy Anabolic reactions - synthesize molecules for cell maintenance, growth and reproduction

Metabolic pathways can be grouped into two paths – catabolism and anabolism

Catabolism is characterized by oxidation reactions and by release of free energy which is transformed to ATP. Anabolism is characterized by reduction reactions and by utilization of energy accumulated in ATP molecules.Catabolism and anabolism are tightly linked together by their coordinated energy requirements: catabolic processes release the energy from food and collect it in the ATP; anabolic processes use the free energy stored in ATP to perform work.

Anabolism and catabolism are coupled by energy

• Multiple-step pathways permit control of energy input and output

• Catabolic multi-step pathways provide energy in smaller stepwise amounts

• Each enzyme in a multi-step pathway usually catalyzes only one single step in the pathway

• Control points occur in multistep pathways

Metabolism Proceeds by Discrete StepsSingle-step vs multi-step

pathways

A multistep enzyme pathway releases energy in smaller amounts that can be used by the cell

• Metabolism is highly regulated to permit organisms to respond to changing conditions

• Most pathways are irreversible

• Flux - flow of material through a metabolic pathway which depends upon:

(1) Supply of substrates(2) Removal of products(3) Pathway enzyme activities

Metabolic Pathways Are Regulated

Levels of Metabolism Regulation

1. Nervous system.2.Endocrine system.3.Interaction between organs.4.Cell (membrane) level.5.Molecular level

• Product of a pathway controls the rate of its own synthesis by inhibiting an early step (usually the first “committed” step (unique to the pathway)

Feedback inhibition

• Metabolite early in the pathway activates an enzyme further down the pathway

Feed-forward activation

Stages of metabolismCatabolism

Stage I. Breakdown of macromolecules (proteins, carbohydrates and lipids to respective building blocks.

Stage II. Amino acids, fatty acids and glucose are oxidized to common metabolite (acetyl CoA)

Stage III. Acetyl CoA is oxidized in citric acid cycle to CO2 and water. As result reduced cofactor, NADH2 and FADH2, are formed which give up their electrons. Electrons are transported via the tissue respiration chain and released energy is coupled directly to ATP synthesis.

Glucose

Pyruvate

Acetyl Co AFatty Acids

Amino Acids

Citric acid cycle supplies

NADH and FADH2 to the

electron transport

chain

Reduced coenzymes NADH and FADH2 are formed in matrix from: (1) Oxidative decarboxilation of pyruvate to acetyl CoA(2) Aerobic oxidation of acetyl CoA by the citric acid cycle(3) Oxidation of fatty acids and amino acids

The NADH and FADH2 are energy-rich molecules because each contains a pair of electrons having a high transfer potential.

The reduced and oxidized forms of NAD

The reduced and oxidized forms of FAD

Electrons of NADH or FADH2 are used to reduce molecular oxygen to water.

A large amount of free energy is liberated.

The electrons from NADH and FADH2 are not transported directly to O2 but are transferred through series of electron carriers that undergo reversible reduction and oxidation.

The flow of electrons through carriers leads to the pumping of protons out of the mitochondrial matrix.

The resulting distribution of protons generates a pH gradient and a transmembrane electrical potential that creates a protonmotive force.

ATP is synthesized when protons flow back to the mitochondrial matrix through an enzyme complex ATP synthase.

The oxidation of fuels and the phosphorylation of ADP are coupled by a proton gradient across the inner mitochondrial membrane.Oxidative phosphorylation is the process in which ATP is formed as a result of the transfer of electrons from NADH or FADH2 to O2 by a series of electron carriers.

OXIDATIVE PHOSPHORYLATION IN EUKARYOTES TAKES PLACE IN MITOCHONDRIATwo membranes:outer membrane inner membrane (folded into cristae) Two compartments: (1) the intermembrane space (2) the matrix

• Inner mitochondrial membrane: Electron transport chainATP synthase

• Mitochondrial matrix:Pyruvate dehydrogenase complexCitric acid cycleFatty acid oxidation

Location of mitochondrial complexes

The outer membrane is permeable to small molecules and ions because it contains pore-forming protein (porin).

The inner membrane is impermeable to ions and polar molecules. Contains transporters (translocases).

THE ELECTRON TRANSPORT CHAINSeries of enzyme complexes (electron carriers) embedded in the inner mitochondrial membrane, which oxidize NADH2 and FADH2 and transport electrons to oxygen is called respiratory electron-transport chain (ETC).

The sequence of electron carriers in ETC

cyt bNADH FMN Fe-S Co-Q Fe-S cyt c1 cyt c cyt a cyt a3 O2

succinate FAD Fe-S

High-Energy Electrons: Redox Potentials and Free-Energy Changes

In oxidative phosphorylation, the electron transfer potential of NADH or FADH2 is converted into the phosphoryl transfer potential of ATP.

Phosphoryl transfer potential is G°' (energy released during the hydrolysis of activated phos-phate compound). G°' for ATP = -7.3 kcal mol-1

Electron transfer potential is expressed as E'o, the (also called redox potential, reduction potential, or oxidation-reduction potential).

E'o (reduction potential) is a measure of how easily a compound can be reduced (how easily it can accept electron).

All compounds are compared to reduction potential of hydrogen wich is 0.0 V.

The larger the value of E'o of a carrier in ETC the better it functions as an electron acceptor (oxidizing factor).

Electrons flow through the ETC components spontaneously in the direction of increasing reduction potentials.

E'o of NADH = -0.32 volts (strong reducing agent)E'o of O2 = +0.82 volts (strong oxidizing agent)

cyt bNADH FMN Fe-S Co-Q Fe-S cyt c1 cyt c cyt a cyt a3 O2

succinate FAD Fe-S

Important characteristic of ETC is the amount of energy released upon electron transfer from one carrier to another.

This energy can be calculated using the formula:

Go’=-nFE’o

n – number of electrons transferred from one carrier to another; F – the Faraday constant (23.06 kcal/volt mol); E’o – the difference in reduction potential between two carriers.

When two electrons pass from NADH to O2 :

Go’=-2*96,5*(+0,82-(-0,32)) = -52.6 kcal/mol

Components of electron-transport chain are arranged in the inner membrane of mitochondria in packages called respiratory assemblies (complexes).

THE RESPIRATORY CHAIN CONSISTS OF FOUR

COMPLEXES

cyt bNADH FMN Fe-S Co-Q Fe-S cyt c1 cyt c cyt a cyt a3 O2

succinate FAD Fe-S

I

III

II

IV

I

II

III IV

Complexes I-IV

• Mobile coenzymes: ubiquinone (Q) and cytochrome c serve as links between ETC complexes

• Complex IV reduces O2 to water

Transfers electrons from NADH to Co Q (ubiquinone) Consist of: - enzyme NADH dehydrogenase (FMN - prosthetic group) - iron-sulfur clusters. NADH reduces FMN to FMNH2. Electrons from FMNH2 pass to a Fe-S clusters. Fe-S proteins convey electrons to ubiquinone. QH2 is formed.

Complex I (NADH-ubiquinone oxidoreductase)

The flow of two electrons from NADH to coenzym Q leads to the pumping of four hydrogen ions out of the matrix.

Complex II (succinate-ubiquinon oxidoreductase) Transfers electrons from succinate to Co Q.

Form 1 consist of: - enzyme succinate dehydrogenase (FAD – prosthetic group) - iron-sulfur clusters. Succinate reduces FAD to FADH2. Then electrons pass to Fe-S proteins which reduce Q to QH2Form 2 and 3 contains enzymes acyl-CoA dehydrogenase (oxidation of fatty acids) and glycerol phosphate dehydrogenase (oxidation of glycerol) which direct the transfer of electrons from acyl CoA to Fe-S proteins.

Complex II does not contribute to proton gradient.

Ubiquinone Q: - lipid soluble molecule, - smallest and most hydrophobic of all the carriers - diffuses within the lipid bilayer - accepts electrons from I and II complexes and passes them to complex III.

All electrons must pass through the ubiquinone (Q)-ubiquinole (QH2) pair.

Complex III (ubiquinol-cytochrome c oxidoreductase)Transfers electrons from ubiquinol to cytochrome c. Consist of: cytochrome b, Fe-S clusters and cytochrome c1. Cytochromes – electron transferring proteins containing a heme prosthetic group (Fe2+ Fe3+).

Oxidation of one QH2 is accompanied by the translocation of 4 H+ across the inner mitochondrial membrane. Two H+ are from the matrix, two from QH2

Complex IV (cytochrome c oxidase)

Transfers electrons from cytochrome c to O2. Composed of: cytochromes a and a3. Catalyzes a four-electron reduction of molecular oxygen (O2) to water (H2O): O2 + 4e- + 4H+ 2H2O

Translocates 2H+ into the intermembrane space

The four protons used for the production of two molecules of water come from the matrix.

The consumption of these four protons contributes to the proton gradient.

Cytochrome c oxidase pumps four additional protons from the matrix to the cytoplasmic side of the membrane in the course of each reaction cycle (mechanism under study).

Totally eight protons are removed from the matrix in one reaction cycle (4 electrons)

Cellular Defense Against Reactive Oxygen Species

If oxygen accepts four electrons - two molecules of H2O are produced

single electron - superoxide anion (O2.-)

two electrons – peroxide (O22-).

O2.-, O2

2- and, particularly, their reaction products are harmful to cell components - reactive oxygen species or ROS.

DEFENSEsuperoxide dismutase (manganese-containing version in mitochondria and a copper-zinc-dependent in cytosol) O2

.- + O2.- + 2H+ = H2O2 + O2

catalaseH2O2 + H2O2 = O2 + 2 H2O

antioxidant vitamins: vitamins E and C reduced glutathione

A PROTON GRADIENT POWERS THE SYNTHESIS OF ATP

The transport of electrons from NADH or FADH2 to O2 via the electron-transport chain is exergonic process:NADH + ½O2 + H+ H2O + NAD+ FADH2 + ½O2 H2O + FAD+ Go’ = -52.6 kcal/mol for NADH -36.3 kcal/mol for FADH2

How this process is coupled to the synthesis of ATP (endergonic process)? ADP + Pi ATP + H2O Go’=+7.3 kcal/mol

• Proposed by Peter Mitchell in the 1960’s (Nobel Prize, 1978)

• Chemiosmotic theory: electron transport and ATP synthesis are coupled by a proton gradient across the inner mitochondrial membrane

Mitchell’s postulates for chemiosmotic theory1. Intact inner mitochondrial membrane is required 2. Electron transport through the ETC generates a proton

gradient3. ATP synthase catalyzes the phosphorylation of ADP in a

reaction driven by movement of H+ across the inner membrane into the matrix

The Chemiosmotic Theory

As electrons flow through complexes of ETC, protons are translocated from matrix into the intermembrane space.

The free energy stored in the proton concentration gradient is tapped as protons reenter the matrix via ATP synthase.

As result ATP is formed from ADP and Pi.

++ + ++ +

----

Overview of oxidative phosphorylation

REGULATION OF OXIDATIVE PHOSPHORYLATION

Coupling of Electron Transport with ATP SynthesisElectron transport is tightly coupled to phosphorylation.

ATP can not be synthesized by oxidative phosphorylation unless there is energy from electron transport.

Electrons do not flow through the electron-transport chain to O2 unless ADP is phosphorylated to ATP.

Important substrates: NADH, O2, ADP

Intramitochondrial ratio ATP/ADP is a control mechanism

High ratio inhibits oxidative phosphorylation as ATP allosterically binds to a subunit of Complex IV

The most important factor in determining the rate of oxidative phosphorylation is the level of ADP. The regulation of the rate of oxidative phosphorylation by the ADP level is called respiratory control

Respiratory control

Uncoupling of Electron Transport with ATP SynthesisUncoupling of oxidative phosphorylation generates heat to maintain body temperature in hibernating animals, in newborns, and in mammals adapted to cold.

Brown adipose tissues is specialized for thermogenesis.

Inner mitochondrial membrane contains uncoupling protein (UCP), or thermogenin.

UCP forms a pathway for the flow of protons from the cytosol to the matrix.

• Uncouplers are lipid-soluble aromatic weak acids• Uncouplers deplete proton gradient by transporting

protons across the membrane

Uncouplers

2,4-Dinitrophenol: an uncoupler• Because the negative charge is delocalized over the ring,

both the acid and base forms of DNP are hydrophobic enough to dissolve in the membrane.

Specific inhibitors of electron transport are invaluable in revealing the sequence of electron carriers.

Rotenone and amytal block electron transfer in Complex I.Antimycin A interferes with electron flow thhrough Complex III. Cyanide, azide, and carbon monoxide block electron flow in Complex IV.

ATP synthase is inhibited by oligomycin which prevent the influx of protons through ATP synthase.

Specific inhibitors of electron transport chain and ATP-synthase

Translocation of 3H+ required by ATP synthase for each ATP produced

1 H+ needed for transport of Pi.

Net: 4 H+ transported for each ATP synthesized

For NADH: 10 H+/ 4H+) = 2.5 ATPFor FADH2: 6 H+/ 4 H+ = 1.5 ATP

ATP Yield

Ten protons are pumped out of the matrix during the two electrons flowing from NADH to O2 (Complex I, III and IV).

Six protons are pumped out of the matrix during the two electrons flowing from FADH2 to O2 (Complex III and IV).

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