Mitochondrial Electron Mitochondrial Electron TransportTransport

• The cheetah, whose capacity for aerobic metabolism makes it one of the fastest animals

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 ChangesIn 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 b

NADH 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

The energy is released not in a single step of electron transfer but in incremental amount at each complex.

26.8

Energy released at three specific steps in the chain is collected in form of transmembrane proton gradient and used to drive the synthesis of ATP.

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.

matrix

NADH-Q oxidoreductase - an enormous enzyme consisting of 34 polypeptide chains. L-shaped (horizontal arm lying in the membrane and a vertical arm that projects into the matrix).

FMNNADH

Iron ions in Fe-S complexes cycle between Fe2+ or Fe3+ states.

Iron-sulfur clusters contains two or four iron ions and two or four inorganic sulfides. Clusters are coordinated by four cysteine residues.

Fe-S

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 QH2

Form 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

Q-cytochrome c oxidoreductase is a dimer.

Each monomer contains 11 subunits.

Q-cytochrome c oxidoreductase contains three hemes: two b-type hemes within cytochrome b, and one c-type heme within cytochrome c1.

Enzyme also contains an iron-sulfur protein with an 2Fe-2S center.

Q cycletwo molecules of QH2 are oxidized to form two

molecules of Q, one molecule of Q is reduced to QH2, two molecules of cytochrome c are reduced, four protons are released on the cytoplasmic side, two protons are removed from the mitochondrial matrix

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

Cytochrome c oxidase consists of 13 subunits and contains two hemes (two iron atom) and three copper ions, arranged as two copper centers.

The Catalytic Cycle of Cytochrome c Oxidise

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.

DEFENSE

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

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

catalase

H2O2 + H2O2 = O2 + 2 H2O

antioxidant vitamins: vitamins E and C reduced glutathione