Flavin mononucleotide FMNFlavin adenine dinucleotide FADRiboflavin

(1)

Development of Complex Curricula for Molecular Bionics and Infobionics Programs within a consortial* framework**

Consortium leader

PETER PAZMANY UNIVERSITY Consortium members

SEMMELWEIS UNIVERSITY, DIALOG CAMPUS PUBLISHER

The Project has been realised with the support of the European Union and has been co-financed by the European Social Fund ***

**Molekuláris bionika és Infobionika Szakok tananyagának komplex fejlesztése konzorciumi keretben

PETER PAZMANY UNIVERSITY

SEMMELWEIS UNIVERSITY

(2)

Semmelweis University

BIOCHEMISTRY

THE ROLE OF MITOCHONDRIA IN BIOENERGETICS

(BIOKÉMIA )

(A MITOKONDRIUMOK SZEREPE A BIOENERGETIKÁBAN )

TRETTER LÁSZLÓ

http://semmelweis-egyetem.hu/

(3)

Biochemistry:

The role of mitochondria in bioenergetics

Energy Relationships between Energy Production (Catabolism) and

Energy Utilization (Anabolism)

CATABOLISM

Energy is released From:

Carbohydrates M Lipids M

Proteins M

ANABOLISM Energy is used For:

Motion, locomotion M Transport processes, M Thermogenesis M

Synthesis of

macromolecules M ATP

NADH NADPH

ADP+P_i

(4)

Biochemistry:

The role of mitochondria in bioenergetics

Introduction to mitochondria The citric acid cycle

The electron transport chain, components, function

Mitochondrial ATP production, the chemiosmotic mechanism Communication between mitochondria and the

extramitochondrial environment

Biochemistry: Mitochondria in bioenergetics

Introduction to mitochondria

http://semmelweis- egyetem.hu/

Electron microscopic

picture of a mitochondrion

Mitochondrion^DEF: Intracellular organelle responsible for the aerobic ATP production.

(6)

Biochemistry: Mitochondria in bioenergetics

Introduction to mitochondria

Pre 1900

First descriptions of mitochondria in cells; speculations about them as bacteria

1930's

1930 Formulation of urea and TCA cycle by H. Krebs

1950's

1950 Identification of mitochondria by electron microscopy (Palade, Sjostrand)

1950s Mitochondria as site of fatty acid oxidation (Lehninger, Kennedy) Mitochondria as site of respiration and oxi.

phosphorylation

1953 Discovery of cytoplasmic inheritance in yeast (Ephrussi, Slonimski)

1955 Definitive respiratory chain analysis (Chance and Williams, Ernster)

1960's & 70's

1960 Characterization of the main components of respiratory chain and ATP synthaser. Description of mobile electron carriers

1961Discovery of mtDNA (M.M.K. and S. Nass)

1980's

1980s A mtDNA sequenching Description of mt protein synthesis.

In vitro mitochondrial protein transport mtDNA replication characterization.

1981 Complete sequence of mammalian DNA 1988 First mitochondrial disease description.

1990's

1990s First sequences of plant mtDNA

1990s Transformation of mtDNA in yeast (introduction of DNA by microprojectiles)

1997 Mitochondriumok és apoptósis: bcl2, cytochrome c; a PTPty

1997 Direct demonstration of rotation of ATP synthase

2000

1995-2000 Crystal structures of complex III, complex IV, F1 ATPsynthase

2000 The Mitochondria Research Societyand the

Mitochondrial chronology

(7)

Biochemistry: Mitochondria in bioenergetics

ENZYMES IN MITOCHONDRIAL COMPARTMENTS

OUTER MEMBRANE INTERMEMBRANE SPACE

MAO (monoamine oxidase) Adenylate kinase,

Kinurenin hidroxilase Nukleoside diphospho kinase NADH cyt c reductase

Carnitine acyl transferase I Porin

INNER MEMBRANE MATRIX

Electron transport chain Citric acid cycle enzymes

ATP synthase Enzymes of beta oxidation

Transporters Pyruvate dehydrogenase

Glutamate dehydrogenase

(8)

Biochemistry: Mitochondria in bioenergetics

PARTICIPATION OF MITOCHONDRIA IN METABOLISM MAJOR PATHWAYS

TCA cycle - universal

Beta oxidation – most of the energy is derived from fatty acid oxidation

Urea cycle – essential for the Nitrogen metabolism

δ−ALA synthesis, porphyrins, cytochromes

Pyrimidin synthesis – dihydroorotate dehydrogenase Cyt P450 – OH C_20,C₂₂

- side chain cleavage - 11β, 18β OH

steroid hormone synthesis

}

(9)

Biochemistry:

Mitochondria in bioenergetics/The citric acid cycle

Learning objectives:

At the end of the presentation students could be able:

•To understand the cyclic character of the tricarboxylic acid cycle

•To understand the bioenergetic importance of the cycle

•To understand the meaning of the regulation of the cycle

(10)

Biochemistry:

Citric acid cycle^DEF: is the final common pathway for the

oxidation of carbohydrates, lipids and proteins. Citric acid cycle generates reducing equivalents for the terminal oxidation.

Acetyl-CoA

C₂ CoA

Oxaloacetate C₄

Citrate C₆

CO₂ CO₂

Synonyms of citric acid cycle:

Krebs cycle

Tricarboxilic acid cycle (TCA cycle) Szent Györgyi-Krebs cycle

(11)

Biochemistry:

Characteristics of citric acid cycle

Acetyl-CoA is combined with oxaloacetate 2 molecules of CO₂ are formed

Oxaloacetate is regenerated

During oxidation reducing equivalents are formed

Reducing equivalents enter into the respiratory chain, ATP is generated in the oxidative phosphorylation

Hypoxia inhibits the cycle because oxygen is the final oxidant of reducing equivalents

Enzymes of the cycle are located in the mitochondrial matrix; freely or attached to the mitochondrial inner membrane

(12)

Biochemistry:

Reactions of the citric acid cycle

Acetyl-CoA

Compounds with red: inhibitors with green: activators

(13)

Biochemistry:

Chemical reactions in the citric acid cycle (enzyme, type of reaction) Citrate synthase: condensation

Aconitase: dehydration, hydration

Isocitrate dehydrogenase: oxidative decarboxylation

Alpha ketoglutarate dehydrogenase: oxidative decarboxylation Succinyl-CoA synthase: substrate level phosphorylation

Succinate dehydrogenase: dehydrogenation Fumarase:hydration

(14)

Biochemistry:

Regulation of the citric acid cycle

Enzyme Inhibitor Activator

Citrate synthase NADH, succinyl-CoA, citrate, ATP

ADP

Isocitrate dehydrogenase ATP, NADH Ca²⁺, ADP

Alpha ketoglutarate dehydrogenase

ATP, NADH, succinyl- CoA

Ca²⁺

Succinate dehydrogenase oxaloacetate

There is no phosphorylation/dephosphorylation in the cycle Local intermediates concentrations regulate the flux.

(15)

Biochemistry:

Mitochondria in bioenergetics/The citric acid cycle Entry of glucose-derived carbons into the cycle Connection between glycolysis and the TCA cycle:

pyruvate dehydrogenase complex

Oxidative decarboxylation of pyruvate

Pyruvate

Acetyl lipoamide Hydroxyethyl TPP

Coenzymes: TPP HS-CoA

(16)

Biochemistry:

+ +

+

- -

-

- - Regulation of PDH complex

Phosphorylated form: inactive Dephosphorylated form: active

Activators: Ca²⁺, pyruvate, ADP, NAD⁺ Inhibitors:, acetyl-CoA, ATP, NADH

(17)

Biochemistry:

Summary of oxidation of pyruvate on PDH complex + citric acid cycle

NADH produced 4

FADH produced 1

Subst. level phosph. 1 CO₂ produced 3

http://www.youtube.com/watch?v=A1DjTM1qnPM&feature=related

Click in the “show” mode for the video about the citric acid cycle and the respiratory chain

(18)

Biochemistry:

Mitochondria in bioenergetics/Electron transport chain Overview of the electron transport chain

(19)

Biochemistry:

Mitochondria in bioenergetics/Electron transport chain

Learning objectives

To understand and to reproduce the flow of electrons through the respiratory chain

To combine the knowledge about the respiratory chain with Mitchell’s postulates

To understand the effect of respiratory chain inhibitors on the oxidoreduction state of the individual chain components

(20)

Biochemistry:

Mitochondria in bioenergetics/Electron transport chain

Electron transport chain^DEF: a sequence of electron-carrying proteins that transfers electrons from respiratory substrates to molecular oxygen in aerobic cells.

Synonyms: respiratory chain, electron transfer chain Components: small molecular mass components

- NAD/NADH, - FAD/FADH₂ - FMN/FMNH₂

- iron-sulphur centers - heme groups

- Coenzyme Q macromolecules (proteins)

}

prosthetic groups of proteins

(21)

Biochemistry:

Respiratory Chain Components Localization Prosthetic

groups

Function

NADPH / NADP(almost 100% reduced) matrix space (separate pool in the cytosol) - mobile carrier

energy-linked transhydrogenaseNADPH + NAD⁺

=> NADH + NADP⁺

Integrant membrane protein none proton pump

2H⁺/2e^-1

NADH / NAD(less than 30% reducedt) matrix space (separate cytosolic pool) - mobile carrier

NADH dehydrogenase (complex 1)

membrane spanning multi-subunit protein non-heme iron

& FMN

proton pump 4H⁺/2e^-1

succinate dehydrogenase

(complex 2) membrane spanning multi-subunit protein non-heme iron

& FAD

no proton pumping

ubiquinol / ubiquinone lipidsoluble - mobile carrier

ubiquinol:cytochrome c reductase (complex 3) membrane spanning multi-subunit protein non-heme iron, heme b & heme c₁ proton pump 4H⁺/2e^-1

cytochrome c (ferrous / ferric)

inter-membrane space heme c mobile carrier

cytochrome c oxidase(complex 4) membrane spanning multi-subunit protein copper, heme a

& heme a₃

proton pump 2H⁺/2e^-1[

F0 / F1 ATPase

(ATP synthetase) (complex V.)

membrane spanning multi-subunit protein none proton pump

3H⁺/ ATP

Components of the respiratory chain and oxidative phosphorylation

(22)

Biochemistry:

A NADH, the most important reducing equivalent

λmax=250 nm

Oxidized form (NAD⁺) Reduced form (NADH)

λmax=340 nm

(23)

Biochemistry:

Mitochondria in bioenergetics/Electron transport chain

Isoalloxazine

Flavin mononucleotide FMN

Flavin adenine dinucleotide FAD Riboflavin

Flavines: FAD and FMN

Flavoproteins in the respiratory chain

Complex I FMN

Complex II FAD

Glycerophosphate FAD dehydrogenase

(24)

Biochemistry:

Mitochondria in bioenergetics/Electron transport chain The oxidoreduction of Coenzyme Q

Coenzyme Q: antioxidant Semiquinone: prooxidant

Function: mobile lipophylic electron carrier in the membrane

(25)

Structure of iron-sulfur centers

Localization of iron sulfur proteins Complex I., complex II, complex III,

Biochemistry:

(26)

Biochemistry:

Mitochondria in bioenergetics/Electron transport chain

/

Heme a Heme c Prosthetic groups of cytochromes

Cytochromes^DEF:heme

proteins, electron carriers in respiration and in other oxido-reduction reactions

(27)

Biochemistry:

Mitochondria in bioenergetics/Electron transport chain

Complexes of the electron transport chain, Complex I

Membrane spanning detailed structure

(28)

Biochemistry:

Mitochondria in bioenergetics/Electron transport chain

Complexes of the respiratory chain: complex II

No proton pumping activity

(29)

Biochemistry:

Mitochondria in bioenergetics/Electron transport chain

The flow of electrons in complex III and

the coenzyme Q cycle

Dimeric form

Membrane spanning Proton pumping activity

(30)

Biochemistry:

Mitochondria in bioenergetics/Electron transport chain

The flow of electrons in the complex IV

Membrane spanning Proton pumping, but less proton can be pumped than in

complex I or III.

Water formation on the matrix side

(31)

Biochemistry:

Mitochondria in bioenergetics/Electron transport chain

Respiratory complexes are organized according to their redox potentials

(32)

Biochemistry:

Mitochondria in bioenergetics/Electron transport chain

Summary

http://www.youtube.com/watch?v=KXsxJNXaT7w&feature=related In “show” mode click to the link below: respiratory chain movie Electrons flow in the respiratory chain from NADH to oxygen Respiratory complex I, III, and IV are able to pump protons to the inner membrane space

10 protons are pumped out/NADH oxidation

The stoichiometry of proton pumping 4; 4; 2; per pair of electrons passed through the complex

Respiratory complexes are organized accoding to their redox potentials

(33)

Biochemistry:

Mitochondria in bioenergetics/ Mitochondrial ATP production

Introduction

In mitochondria ATP is synthesized via oxidative phosphorylation.

Oxidative phosphorylation^DEF: oxidation (oxygen consumption, or oxidation of reducing equivalents) is coupled to phosphorylation i.e.

to ATP synthesis. The oxygen consumption thus coupled to ATP synthesis

(34)

Biochemistry:

Mitochondria in bioenergetics/ Mitochondrial ATP production

http://semmelweis- egyetem.hu/

1)The membrane-located ATPase systems of mitochondria and chloroplasts are hydro-dehydration systems with terminal

specificities for water and ATP; and their normal function is to couple reversibly the translocation of protons across the membraneto the flow of anhydro-bond equivalents between water and the couple ATP/(ADP + Pi).

2)The membrane-located oxido-reduction chain systems of mitochondria and chloroplasts catalyse the flow of reducing equivalents, such as hydrogen groups and electron pairs, between substrates of different oxido-reduction potential; and their normal function is to couple reversibly the translocation of protons across the membrane to the flow of reducing equivalentsduring oxido-reduction.

3)There are present in the membrane of mitochondria and chloroplasts substrate-specific exchange- diffusion carrier systemsthat permit the effective reversible transmembrane exchange of anions against OH- and of cations against H+; and the normal normal function of these systems is to regulate the pH and osmotic differential across the membrane, and to permit entry and exit of essential metabolites (e.g., substrates and phosphate acceptor) without collapse of the membrane potential.

4)The systems of postulates 1, 2, and 3 are located in a

specialized coupling membranewhich has a low permeability to protons and to anions and cations generally.

MITCHELL, P. Coupling of phosphorylation to electron and hydrogen transfer by a chemiosmotic type of mechanism. Nature 191:144–148, 1961.

Nobel Prize in Chemistry 1978

Mitchell’s postulates

H⁺ ADP+P_i

ATP reducing

equivalents

H₂O

H

⁺

Ions,

metabolites H⁺

(35)

Biochemistry:

Mitochondria in bioenergetics/ Mitochondrial ATP production

Oxidation–Reduction Potentials of the Mitochondrial Electron Transport Chain Carriers

(36)

Biochemistry:

Energy derived from NADH oxidation NADH + H⁺ + ½ O₂

ΔG^o’= - NFΔE^o’ n= number of electrons, F=Faraday number

=2(96.5 kJ/V*mol)(1.14 V) ΔE^o’=st. redox potential difference

=220 kJ/mol between NAD/NADH and oxygen/water pairs

As a consequence of proton pumping activity membrane potential (matrix negative) and pH difference is formed across the two sides of the membrane.

Proton motive force (pmf) ΔμH⁺=ΔΨm-60ΔpH (mV)

Energetics of ATP synthesis

(37)

Biochemistry:

How much energy is released when one proton moves from the intermembrane space to the matrix?l

In energized mitochondria.. ΔpH~0.75;

ΔΨ~150-200 mV

Proton motive force: ΔG=2.3RTΔpH + FΔΨ=5.70kJ/mol*ΔpH + (96.5kJ/V*mol)ΔΨ≅20 kJ/mol Energy required to pump out one proton

cca 20 kJ/mol

10 protons would cost about ΔG~200 kJ

However, the oxidation of NADH releases 220 kJ. The efficacy of proton pumping about 200/220 (Energy derived from NADH oxidation

NADH + H+ + ½ O2 ΔGo’= - NFΔEo’

=2(96.5 kJ/V*mol)(1.14 V)

=220 kJ/mol)

Energetics of ATP synthesis

(38)

Biochemistry:

F₁F_o ATP-ase produces ATP

from the energy of proton gradient

http://www.youtube.com/watch?

v=uOoHKCMAUMc

Click to the link below to play the movie about the ATP

synthesis

(39)

Biochemistry:

Functional characteristics of the ATP synthase molecule

(40)

Biochemistry:

The molecular mechanism of ATP synthesis

(41)

Biochemistry:

Mitochondria in bioenergetics/ Mitochondrial ATP production

Cytosol Inner membrane Matrix

ADP^3-

ATP^4-

H₃PO₄^- H⁺

Phosphate carrier Adenine Nucleotide translocase ANT is electrogenic: there is a

net movement of one negative charge from the matrix to the intermembrane space

ANT can be reversed. In that case ATP from the cytosol enters into the mitochondria

Electrogenic transport^DEF: A transport is defined as electrogenic if during the transport process there is a net charge movement. As a consequence of it a current is generated

Transports essential for the oxidative

phosphorylation

Phosphate carrier is electroneutral

(42)

Biochemistry:

Mitochondria in bioenergetics/ Mitochondrial ATP production

How much energy is required to synthesize one mol ATP?

How much energy is liberated during the hydrolyzis of 1 mol ATP?

ΔG= ΔGo’ + RTln[ADP][Pi]/[ATP]

ΔG=-30.5kJ/mol + 8.315*298

=-54.7 kJ/mol muscle cell

ΔG=-51.8 kJ/mol Red Blood Cell

2 protons are not enough (2x20=40 kJ less than 50-55kJ) 3 proton is more than enough but:

Because of the ATP/ADP exchanger one extra proton is required Total cost: 4 protons/ATP

10 protons/NADH could form 2.5 mol ATP/NADH oxidation

(43)

Biochemistry:

Mitochondria in bioenergetics/ Mitochondrial ATP production P/O ratio^DEF: P/O or ATP/O ratio (where O refers

to an oxygen atom) is the number of ATP molecules formed when one oxygen atom is reduced to water

P/O ratio for NADH oxidation: 10 protons are pumped during oxidation 1 NADH will reduce 1 oxygen atom

10 protons are enough to synthesize 2.5 mol ATP-t

How much is the P/O ratio during the oxidation of succinate?

Succinate oxidation does not involve proton pumping at complex I.

Complex II is unable to pump protons.

The number of proton pumped 4(complex III) +2(complex IV)=6 6 protons produce 1.5 mol ATP

P/O_succinate=6/4=1.5

(44)

Biochemistry:

Mitochondria in bioenergetics/ Mitochondrial ATP production

Energy balance of glycolysis+citric acid cycle

Site ATP substrate

level

phosphorylation

ATP from oxidation of reducing

equivalent

sum

Glycolysis P-glycerate kinase Pyruvate kinase

1 1

2 Gliceraldehyde-P

dehydrogenase

2x2.5 5

Pyruvate dehydrogenase

2x2.5 5

Citric acid cycle ICDH 2x2.5 5

α-KGDH 2x2.5 5

Succinate thiokinase

2 2

Succinate dehydrogenase

2x1.5 3

Malate

dehydrogenase

2x2.5 5

Summary 4 28 32

(45)

Biochemistry:

Mitochondria in bioenergetics Mitochondrial ATP production

Uncoupling of oxidative phosphorylation

Normal Uncoupled

ADP+Pi

High energy protons Low energy protons

X

uncoupler

Protons cross the ATP synthase and drive ATP synthesis

Protons are carried by the protonophor

(uncoupler).Protons cross the membrane without using ATP synthase. Proton gradient is dissipated without ATP synthesis

(46)

Biochemistry:

Mitochondria in bioenergetics/ Mitochondrial ATP production

Experiments proving the chemiosmotic ATP generation

INNER MEMBRANE

ULTRASOUND

INSIDE OUT

SUBMITOCHONDRIAL PARTICLES

MECHANICAL SHAKING

MEMBRANE VESICLES ELECTRON TRANSPORT + ATP SYNTHESIS -

F₁ PARTICLES

ELEKTRON TRANSPORT – ATP SYNTHEZIS -

ATP-ASE ACTIVITY + RECONSTRUCTION

EL. TRPORT + ATP SYNTHESIS +

F₁

F_o

NADH+O₂ ADP+Pi

NAD+H₂O ATP

(47)

Biochemistry:

Δ[H⁺]

pH electrode O₂injector

O₂ injection

Mitochondria respire

(oxygen consumption and proton pumping activity)

OXYGEN DECREASES,

PROTON PUMPING ACTIVITY DECREASES

(48)

Biochemistry:

The role of proton motive force in ATP synthesis 1961

Peter Mitchell

pH:7.5 pH 4.0 pH 4.0

pH:7.5 pH 4.0

Equilibration with pH 4.0 buffer

Buffer pH 8.0 +ADP + P_i

ADP+P_i

ADP+P_i ATP

ATP

ATP synthesis

Artifitially constructed pH gradient

(49)

Biochemistry:

Mitochondria in bioenergetics/ Mitochondrial ATP production

Liposomes

H⁺ H⁺ H⁺ H⁺ H⁺

H⁺

H⁺ H⁺ H⁺ H⁺ H⁺

H⁺ H⁺ H⁺

H⁺ H⁺

Bakteriorhodopsin

ADP+P_i

ATP F

0

F₁

H⁺

Artifitial vesicles

From purified phospholipids

Protons pumped by bacteriorhodopsin The role of proton motive force in ATP synthesis

(50)

Biochemistry:

Mitochondria in bioenergetics/ Mitochondrial ATP production

The essence of oxidative phosphorylation

MEMBRANE

- - - -

+ + + + + + + ATP

ADP+P_I

H+

O₂ H₂O

(51)

Biochemistry:

Mitochondria in bioenergetics/ Mitochondrial ATP production

Summary

The energy of substrate oxidation forms proton gradient High energy protons are used for ATP synthesis

3 protons are necessary to cover the actual expenses of ATP synthesis However, besides the respiratory chain and the ATP synthase ANT and Pi carrier are also essential for the oxidative phosphorylation

ANT is an electrogenic carrier, every catalytic cycle decreases membrane potential

The electrogenic property of ANT adds a further proton to the costs of ATP synthesis, so 4H⁺s are needed to synthesize 1 ATP molecule

The P/O ratio for NADH is 2.5 Uncoupling decreases P/O ratio

(52)

Biochemistry: M itochondria in bioenergetics Mitochondrion-cytosol communication

Learning objectives

To understand the essential role of mitochondrion-cytosol communication:

- in the metabolite transport (with special emphasis to the oxidation of glycolytic NADH)

- in the calcium homeostasis. Students can understand the importance of various Ca²⁺ uptake and Ca²⁺ release pathways

The importance of mPTP opening can not be overestimated in pathology.

Students could get an impression about the nonphysiological conditions associated with mitochondria.

The unique properties of mtDNA and the flow of information between the

(53)

Biochemistry: M itochondria in bioenergetics Mitochondrion-cytosol communication

Phosphate

Malate

Pyruvate

OH^- Malate

Citrate

Malate

Alpha-ketoglutarate malate – α-KG carrier Dicarboxylate carrier

Tricarboxylate carrier

Monocarboxylate carrier Mitochondrial substrate carriers

(54)

Biochemistry: M itochondria in bioenergetics Mitochondrion-cytosol communication

Shuttle of reducing equivalents

Glycolysis produces NADH. NADH could reduce pyruvate to

lactate, however lactate formation prevents entry of pyruvate into the matrix, thus oxidative phosphorylation is inhibited.

Cytosolic NADH accordingly should be oxidized in the

mitochondria, but mitochondrial inner membrane is impermeable to NAD⁺ and NADH.

Those mechanisms which help the oxidation of cytosolic NADH in the mitochondria are called “shuttles”.

Glycerophosphate dehydrogenase localization: inner membrane, outer surface. Redox potential between complex I and III (like complex II) thus 6H⁺/glycerophosphate oxidation are pumped out.

(55)

Biochemistry: M itochondria in bioenergetics Mitochondrion-cytosol communication

Malate

Glutamate

Aspartate – glutamate carrier Malate malate – α-KG carrier

The malate-aspartate shuttle

cytosol matrix

IM

glyceraldehyde3-P

G L Y C O L Y S I S glucose

pyruvate

1,3 bisphosphoglycerate

Pi NAD

NADH

α-Ketoglutarate OA

Aspartate

OA

α-KG

Aspartate Glutamate

NAD NADH NADH NAD

GOT

R E S P I R A T O R Y C H

(56)

Biochemistry: M itochondria in bioenergetics Mitochondrion-cytosol communication

glyceraldehyde3-P

G L Y C O L Y S I S glucose

pyruvate

1,3 bisphosphoglycerate

Pi _NAD

NADH

O||

CH| HCOH|

CH₂O P

CH₂OH C=O| CH| ₂O P CH₂OH HCOH|

CH| ₂O P

O||

CO∼ ^P

HCOH| CH| ₂O P

Glycerol 3-P

Dihyroxyacetone P

FAD

FADH₂

The glycerophosphate shuttle

Glycerol-P dehydrogenase

Cytosol Inner membrane Matrix

(57)

Biochemistry: M itochondria in bioenergetics Mitochondrion-cytosol communication

Mitochondrial Ca²⁺ homeostasis

UNIPORTER

(58)

Biochemistry: M itochondria in bioenergetics Mitochondrion-cytosol communication

time (sec)

0 15 30 45 60 75 90 105 120

Normalized ΔF/F 0

0.0 0.5 1.0

ATP

time (sec)

0 15 30 45 60 75 90 105 120

NormalizedΔF/F 0

0.0 0.5 1.0

ATP FCCP

time (sec)

0 15 30 45 60 75 90 105 120 135 150 165 180 195 210

Normalized ΔF/F 0

0.0 0.5 1.0 1.5

ATP CGP-37157

Mitochondrial Uptake of Ca²⁺

Æ Na

⁺

/Ca

²⁺

-exchange is important

nuclei ~ cytosol (raw) mitochondria (HF)

Æ ΔΨ

_m

dependent Æ

slower kinetics

ATP: purinergic agonist G_q->PLC->IP₃

FCCP: uncoupler

CGP-37157: Na-Ca exch inhib.

(59)

Biochemistry: M itochondria in bioenergetics Mitochondrion-cytosol communication

Mitochondrial Ca²⁺ transporters

(60)

Biochemistry: M itochondria in bioenergetics Mitochondrion-cytosol communication

Ca

²⁺

uniporter

– Ca²⁺elecrtophoretic uptake (charge carried: 2)

– Voltage dept, Ca²⁺ activated – K_m~10μM

– InhibitorsRuthenium red, Ru360 (- )

– Kompetetive inhibitors bound and transported in the channels:

Sr²⁺,Mn²⁺,Ba²⁺

– Allosteric inhibitors: Mg²⁺,Mn²⁺, – Spermin (+)

– ATP>ADP>AMP (-)

– Unknown molecular identity

Rapid uptake mode

– Ca²⁺uptake [Ca²⁺]_o~400nM Transient uptake,

– Reset

– Inhibitor: Ruthenium red, Ru360 (- but at higher conc.)

– ATP activated

– Mg²⁺does not inhibit

(61)

Biochemistry: M itochondria in bioenergetics Mitochondrion-cytosol communication

Na

⁺

-indept Ca

²⁺

-efflux (NICE)

– nH⁺-Ca²⁺ antiporter, n>2 ΔΨ_m required – Ruthenium Red insensitive

– Slow, early saturation

Na

⁺

-dept Ca

²⁺

-efflux (NCE)

– 3Na⁺-Ca²⁺ antiporter – ΔΨ_m required

– Mg²⁺,Sr²⁺,Ba²⁺,Mn²⁺(-)

– Amilorid, trifluoperazin, diltiazem, CGP- 37157 (-)

Ca

²⁺

-cycling

– Could contribute to the fine

(62)

Biochemistry: M itochondria in bioenergetics Mitochondrion-cytosol communication

pyruvate

Ca²⁺ activates TCA cycle

[Ca

²⁺

]

_matrix

Pyruvate dehydrogenase

complex

α-ketoglutarate dehydrogenase

Isocytrate dehydrogenase

+ +

+

[Ca

²⁺

]

_matrix

Pyruvate dehydrogenase

complex

α-ketoglutarate dehydrogenase

Isocytrate dehydrogenase

+ +

+

(63)

Biochemistry: M itochondria in bioenergetics Mitochondrion-cytosol communication

Hypothetical scheme of mitochondrial Permeability Transition Pore (mPTP)

(64)

Biochemistry: M itochondria in bioenergetics Mitochondrion-cytosol communication

Mitochondrial permeability

transition

^DEF

: The mitochondrial permeability transition involves a sudden (and initially reversible)

increase in permeability of the IMM to solutes up to 1.5 kDa. It is commonly defined by its inhibition by cyclosporin A.

(65)

Biochemistry: M itochondria in bioenergetics Mitochondrion-cytosol communication

Regulation of mPTP

The most important mPTP opener is high matrix [Ca

²⁺

] !

– Voltage sensitive: IMM depolarization opens

• gating potential is shifted by agonist (more negative ΔΨm)

• or antagonists (less negative ΔΨm) – Divalent cations:

• [Ca²⁺]_Matrix (+)

• [Mg²⁺]_Matrix , [Mn²⁺]_Matrix, divalent cations outside (-)

– Matrix pH: Alkalization is permissive (>= pH 7.3); OH^-,P_i (+)

– Thiol oxidation: Oxidation (disulphide formation) of ANT (+)

• redox status is in equilibrium with matrix

(66)

Biochemistry: M itochondria in bioenergetics Mitochondrion-cytosol communication

Regulation of mPTP – ANT ligands:

• ADP, bongkrekate (-)

• Atractyloside (+) – Metabolites:

• Glucose and creatine inhibit (-) (action on hexokinase and creatine kinase)

• Coenzyme Q (-)

• Long-chain fatty acids, ceramide and ganglioside GD3 (+)

– Anti- and pro-apoptotic members of the Bcl-2 family.

Multiple conductance state

– Low conductance state for selective permeation of H⁺ or Ca²⁺

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Biochemistry: M itochondria in bioenergetics Mitochondrion-cytosol communication

Metabolic consequences of mPTP opening

Uncoupling of the respiratory chain with collapse of the proton gradient Cessation of ATP synthesis

Matrix Ca²⁺ outflow

Depletion of reduced glutathione Depletion of NADPH

Hypergeneration of O₂^{. –}

Release of intermembrane proteins

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Biochemistry: M itochondria in bioenergetics Mitochondrion-cytosol communication

Physiological function of mPTP

Periodic reversible opening of the permeability transition pore allows for

– release of Ca²⁺ from the mitochondrial matrix, thereby participating in Ca²⁺

homeostasis and/or the generation of Ca²⁺ waves

– The ANT/VDAC couple (and its interacting proteins hexokinase and creatine kinase) may also participate in regulating ATP/ADP transport/synthesis.

Irreversible permeability transition

– triggers mitochondrial autophagy (a process by which cells digest parts of their cytoplasm), apoptosis or necrosis.

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Biochemistry: M itochondria in bioenergetics Mitochondrion-cytosol communication

Physiological uncoupling: brown adipose tissue cold adaptation

UCP-1 protein uncoupling Heat generation

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Biochemistry: M itochondria in bioenergetics Mitochondrion-cytosol communication

The mitochondrial genom

16,569 base pair

7 of 43 subunits of complex I (ND1, 2, 3, 4, 4L, 5, and 6),

1 of 11

subunits of complex III (cytochrome b, cyt b),

3 of 13

subunits of complex IV (COI, II, and III),

2 of 16 subunits

of ATP synthase (ATPase 6 and 8),

+ small and large rRNAs, 22 tRNAs

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Biochemistry: M itochondria in bioenergetics Mitochondrion-cytosol communication

Summary

There is an intensive communication between the mitochondria and the other intracellular compartments.

Metabolite trafficking is essential, because metabolic fuels should enter into the mitochondria, ATP, and waste products have to leave it.

The NADH shuttle systems are particularly important in the oxidation of cytosol-generated NADH.

Mitochondria play an important role in the Ca²⁺ homeostasis. Ca²⁺ can be an important signal for mitochondria to increase energy production, but Ca²⁺ overload could kill mitochondria opening the mPTP.

Although probably the most important mitochondrial task is to supply

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Biochemistry:

Mitochondria in bioenergetics

Recommended literature

Orvosi Biokémia (Ed. Ádám Veronika) Textbook of Biochemistry

Ed. Thomas Devlin 5^th-7th edition

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Biochemistry:

Mitochondria in bioenergetics

Questions

Calculate P/O ratio if only complex IV is in the position to pump protons Which enzymes in the mitochondria are activated by calcium?

How many ATP is produced in the citric acid cycle if NADH is oxidized in the respiratory chain?

What is the effect of uncoupling on the transmembrane pH difference?

What are the transporters for the mitochondrial calcium efflux

What are the well-known functions of the hypothetical constituents of the mPTP?

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Biochemistry:

Mitochondria in bioenergetics

Questions

Which statements are true for the thermodinamics of the respiratory chain?

A: The change of redoxpotential during the oxidation of a NADH is 1.14V

B: The energy of a single proton in energized mitochondria is about 20 KJ/mol C: In order to synthesize 1 ATP molecule we need the energy of 4 protons D: The standard free energy of ATP hydrolysis differs from the actual one E: All of the statements are true

Which of the following enzymes produce NADH in the citroc acid cycle A: Aconitase

B: Glutamate dehydrogenase C: Succinate dehydrogenase D: Malate dehydrogenase E: Citrate synthase

Flavin mononucleotide FMNFlavin adenine dinucleotide FADRiboflavin

BIOCHEMISTRY

THE ROLE OF MITOCHONDRIA IN BIOENERGETICS

TRETTER LÁSZLÓ

Biochemistry:

The role of mitochondria in bioenergetics

Biochemistry:

The role of mitochondria in bioenergetics

Introduction to mitochondria The citric acid cycle

The electron transport chain, components, function

Mitochondrial ATP production, the chemiosmotic mechanism Communication between mitochondria and the

extramitochondrial environment

Table of contents

Biochemistry: Mitochondria in bioenergetics

Introduction to mitochondria

Biochemistry: Mitochondria in bioenergetics

Biochemistry: Mitochondria in bioenergetics

Biochemistry: Mitochondria in bioenergetics

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How much energy is liberated during the hydrolyzis of 1 mol ATP?

=-54.7 kJ/mol muscle cell

2 protons are not enough (2x20=40 kJ less than 50-55kJ) 3 proton is more than enough but:

Because of the ATP/ADP exchanger one extra proton is required Total cost: 4 protons/ATP

10 protons/NADH could form 2.5 mol ATP/NADH oxidation

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