A bit of chemistry Contact Details Free change

Gibb's Free Energy change is a concept which allows to predict if Dr Alexander Galkin a reaction is thermodynamically favorable

A + B ↔ C ∆G<0 – spontaneous reaction →, EXErgonic Microbial energy Email: [email protected] ∆G>0 – no reaction, ENDErgonic 210BBC210 Office: MBC Room 01.442 A + B A + B

Tel: (028) 90972166 CC

Free energy change (ATP) Redox reactions (reduction-oxidation reactions) ∆Go = change of free energy of reaction at standard state conditions at ATP is regarded as a universal source of energy 1M concentration of reactants. But in reality concentrations may vary! occurring in all types. In animals it is produced during the degradation of foodstuff. A + B ↔ C ∆Go>0 – no reaction if we mix A, B and C at concentrations of 1M ([A]=[B]=[C]=1M) → ATP ADP + P i + energy ]C[ × ∆ G 0 +∆=∆ lnG However, if [A] [B] >>[C], real G<0 and × ]B[]A[ reaction will go from left to right →

A + B → A + B Midpoint redox potential ( Eo) is a tendency In particular case of standard 0 1 0 red ox ox red G +∆=∆ lnG +∆= 0G of A to donate electrons conditions ∆G= ∆Go because × ------red 1 1 → - Ared Aox + 2e -→ Electrons transferred from A to B if Box + 2e Bred red ox Eo < Eo Enzymes accelerate the attainment of equilibrium, but not shift it or reverse reaction. Aox /A red Box /B red ∆ Direction of the reaction is defined by G. Some of the biological reactions have Burning (oxidation) of Cells can store energy during ATP can be used to drive any ∆Go>0, but due to the concentration component (in logarithm) ∆G<0. glucose to CO 2 and H 2O glucose oxidation can in form cellular processes produces heat only of ATP

1 and Respiration Reduction of Oxidative phosphorylation (energy conversion) History

+hν ↔↔↔ ↔↔↔ CO 2 + H 2O {CH 2O} + O2 {CH 2O}+ O2 CO 2 + H 2O + energy Photosynthetic Animals or microorganisms W. A. Engelhardt, 1936-39 - measured inorganic and organic phosphate content definition of oxidative phosphorylation Capture of solar energy to use it for reduction Oxidation of food to obtain energy of carbon compounds Warburg vs Thunberg and Keilin - respiratory vs dehydrogenase

Albert Lehninger – 1948 – mitochondria are the site of energy metabolism Animals David Green – 50s, isolation and reconstitution of electron transport chain

- + ↔ Piter Mitchell – energy transduction in membranes Nobel Prize 1978 O2 +4e +4H 2H 2O


Mitochondria – respiring Membranes of mitochondria and bacteria What are mitochondria?

• An intracellular organelle. • There are 100 to 1000s of mitochondria/cell. • All mitochondria come from the mother. • Mitochondria have their own DNA. • Major functions of mitochondria: – Makes energy in the form of ATP.

Also contain its own DNA and its own Endosymbiotic theory transcription/translational system

2 Mitochondrial DNA Locations Double stranded, circular Bacteria

• No introns, 80 - 93% coding gene

• No repeats • Glycolysis • Lack histone and DNA repair mechanism ⇒ – Cytoplasm Cytoplasm Cytoplasm damage, mutations (free radicals)

• 37 gene: 22 tRNA, 2 rRNA & 13 • Krebs’ TCA – Mitochondrial matrix • Oxidative phosphorylation NUCLEOID A dynamic complex that consists of Plasma membrane several copies of mitochondrial DNA and – Inner mitochondrial membrane key maintenance within the organelle.

POLYCISTRONIC A form of gene organization that results ⇒⇒⇒ Compartmentalisation in transcription of an mRNA that codes for multiple gene products, each of which is independently translated from the Mitochondrial matrix mRNA.

Electron transferring coenzyme Krebs cycle (TCA cycle) After glycolysis and TCA cycle 2 Carbon Not much ATP formed Lots of reduced coenzymes

4 Carbon 6 Carbon Per glucose : 10 NADH

2 FADH 2 (!!!) At the same time:

NAD + + 2e - +H + ↔ NADH Reoxidation of NADH releases energy Requires oxygen as oxidant This energy can be used for ATP synthesis Mitochondrial matrix

3 Electron transport chain and ATP Redox centres synthase

Flavin Respiratory chain couples processes of Iron- sulphur centres (FeS-centres) oxidation and ATP synthesis Ubiquinone H+ Cytochromes in

Inner membrane H+ out

Flavin FeS clusters Ubiquinone

2 electrons + 2H + Complex I Complex I Complex II = succinate dehydrogenase from Krebs cycle Complex II = succinate dehydrogenase from Krebs cycle Membrane mobile redox carrier linking Complexes I and II with Complex III Complex III = bc 1 complex Proton-translocating Q-cycle in complex III

Different n for different species ( n=6-10)

Menaquinone and rhodoquinone in some bacteria and plastoquinone in

- + ↔ FAD + 2e +2H FADH 2

Usually serves as intermediate of electron Always transfer one electron at the time transfer between 2e - donor and 1e- acceptor Electron is delocalised No free flavins in a cell !!!

4 Cytochromes Respiratory chain cytochromes

Reduced-oxidised spectra of Cytochrome c Reduced-oxidised spectra ------a ------b ------c mitochondrial membranes

Hugo Theorell Nobel prize 1955 Otto Warburg Nobel Prize 1931 Redox and biological Nature and mode of action of the oxidation Cyt c (Fe 3+ ) + e - ↔ Cyt c (Fe 2+ ) respiratory enzymes

Protein part and haem part containing Fe ion Cytochromes a, b, c … + number As separate proteins (e.g. cytochrome c) or as subunits of enzymatic complexes (Complex II, III, IV) Easy to observe reduction/oxidation as change in optical spectra

Electron transport chain Electron transport chain Structure of respiratory chain

Values of redox potential of respiratory chain components II I III IV V fall downstream from NADH to oxygen.

bc 1 complex

NADH:ubiquinone Succinate oxidoreductase Cytochrome c dehydrogenase oxidase

ATP synthase

5 Complex I (NADH:ubiquinone oxidoreductase)

+ + ↔ NADH + H + 4H in + UQ ↔ + + NAD + UQH 2 + 4H out

Mammalian enzyme : 45 subunits Bacterial enzyme : 14 subunits Flavin = FMN 8 FeS clusters Tightly-bound semiquinones as intermediates of electron transfer

Classical inhibitors : Rotenone, piericidine, MPP +

6 ATP synthase ATP synthase ATP-synthase

Nobel prize 1997 John Walker

Bacterial enzyme : 8 subunits

Proton flow through Fo part is coupled with ATP synthesis in the F 1 part Yoshida Lab http://www.res.titech.ac.jp/~seibutu/ + ~10H per 3 of ATP Kinosita Lab http://www.k2.phys.waseda.ac.jp/Movies.html

ATP synthesis in mitochondria ATP transport ATP synthesis (in mitochondria!)

Matrix Intermembrane ADP Respiration space ATP synthesis in mitochondrial matrix ATP Electron transport Needs to be transported out of H+ chain: Matrix Generates ∆p mitochondria Requires ATP-ADP transporter ATP-ADP transporter Integral membrane protein H+ ATP synthase: Consumes ∆p ATP and ADP transport coupled Inter-membrane space ADP Energy requiring ATP processes in the cell

7 Mitochondrial respiration How to study electron transport chain? Oxidative phosphorylation inhibitors

History: Isolated mitochondria + substrates + oxygen • I – Rotenone – Ubiquinone-like structure Some compounds block oxygen consumption – • II – Oxaloacetate respiration inhibitors – Succinate-like structure

Some compounds stop ATP synthesis but not respiration, • III - Antimycin A they break the link between respiration and ATP – Fungicide and insecticide - - synthesis – uncouplers • IV - Cyanide (CN ), azide (N 3 ), (CO), nitric oxide (NO)

– Similar electronic structures to O 2

Protonmotive force Mitochondrial respiration Oxidative phosphorylation

Respiratory control ratio

H+/2e - stoichiometry of respiratory chain complexes

H+/ATP stoichiometry of ATP synthase

ADP/O ratio – how much ADP can be converted to ATP per molecule of oxygen

Reversibility of reactions = reverse electron transfer

Reactive oxygen species generation

8 Aerobic respiration Aerobic respiration Aerobic respiration (in mammals!)

Glucose (C ) Inner mitochondrial membrane ~1400m 2 →→→ 6 + 2 ADP C6H12 O6 + 6O 2 6CO 2 + 6H 2O 4 NAD Glycolysis We consume ~380 l of oxygen per day 10 NADH ~30 ADP ATP ADP 4 NADH + 2 H + 2 ATP e- ATP turnover ~60 kg/day

Carbon dioxide: 2 CO 2 →→→ 2 Ac CoA (2 C 2) e- 3 × 10 23 H+ per second through ATP Released in Pyruvate AcCoA 2 FADH 2 UQ synthase and Krebs’ TCA 2 FAD e- 4 CO 2 Oxygen: 90% ATP is synthesised during oxidative 2 FADH 2 Required at the end of Krebs’ Cyt c phosphorylation TCA oxidative phosphorylation 2 GDP e- ~30 ATP 2 GTP O2 Glucose: 6 NAD + Oxidative phosphorylation Chemical energy from food 6 NADH + 6 H +

Bacterial energy metabolism Membranes of mitochondria and bacteria What to do if food is scarce?

Live in various environment

Able metabolise different substrates

Can adopt to the changing environment

Some bacteria can grow on low carbon substrates like methanol as its sole source of energy. In P. denitrificans methanol is oxidised in periplasm and electrons are transferred directly to cytochrome oxidase

9 Simplification Bacterial energy metabolism Bypassing Complex I =

NADH dehydrogenase Type 1 bc 1 (III) and cytochrome c oxidase (IV) bo 3 oxidase (quinol oxidase) (eukaryotes) together (in mitochondria ) (E.coli, Burkholderia phymatum) NADH dehydrogenase Type 2 In many bacteria efficiency of respiration (ATP:O ratio) is (E.coli, Rhodobacter capsulatus, etc) P lower than in mitochondria QH 2 More simple machinery of H +/e - transport Q

Bypassing Q N

Shortening or branching of the chain QH 2

Proton translocation No proton translocation (not even a membrane 2QH + O + 4H + ↔ 2Q + 2H O + 4H + protein) 2 2 in 2 out Direct oxidation of quinol by oxygen, bypassing bc complex and + ↔ + 1 NADH + H + Q NAD + QH 2 cyt c + translocation of protons (but with lower efficiency)

Scheme of Respiratory Chains of What to do if the oxygen is absent? Bypassing and simplification mitochondria and bacteria What would be the terminal electron acceptor or where electrons should end up?

bo 3 oxidase (quinol oxidase) (E.coli, Burkholderia phymatum)

bd oxidase (quinol oxidase) (no H + translocation) (E.coli, Klebsiella pneumoniae, Mycobacterium tuberculosis ) ↔ 2QH 2 + O 2 2Q + 2H 2O P. denitrificans nitrate reductase system

Direct oxidation of quinol by oxygen, bypassing bc 1 complex and cyt c + but no (!!!) translocation of protons - → - → → → NO 3 NO 2 NO N2O N2

10 What to do if the oxygen is absent? How to generate proton gradient without actual Fumarate reductase from E.coli What would be the terminal electron acceptor or where electrons should end up? proton translocation? NADH E = -320mV Fumarate reduction in E.coli ↔ o Fumarate + UQH 2 Succinate + UQ

NADH up to 4H +/2e - Oxidation of formiate coupled with Dehydrogenase Very similar to mitochondrial translocated fumarate reduction in E.coli Type 1 succinate dehydrogenase

4 subunits Menaquinone E = -74mV o N

Flavin – FAD Fumarate reductase 3 FeS clusters Cyt b, FeS, FAD

Net reaction: Why use menaquinone + P release 2H at P-side Fumarate Eo= +33mV instead of ubiquinone? consumption of 2H + at N-side

E. coli respiratory chain P. denitrificans respiratory chain What to do if it is very alkaline outside or too much salt?

Ions other than H + can be used. In certain bacteria gradient of Na + is created by special enzymes and can be used by special Na + translocating ATP synthase for ATP synthesis.

Sodium bioenergetics: Halophilic bacteria, Vibrio cholerae, Yersinia pestis

The components in italics are constitutive. The other components are induced at appropriate growth conditions and are unlikely to be all present at once. Continuous boxes indicate integral membrane components; dashed lines The components present depend on the growth conditions. Menaquinone represent periplasmic components. NADH dehydrogenase, succinate replaces ubiquinone under anaerobic conditions. Many of the enzymes dehydrogenase, ubiquinol cytochrome c oxidoreductase and aa 3 oxidase (e.g. reductase) have their active sites at the periplasm. correspond to mitochondrial complexes I-IV.

11 Flagellum = whip How does it work?

Energy of proton gradient can be converted in to mechanical movement (E.coli, Helicobacter pylori, Salmonella typhimurium, Halobacterium ) ~10 3 rpm = 300 Hz = 300 revolutions per second

Using two fuels How to use light ? Bacteriorhodopsin Bacteriorhodopsin is a protein from halobacteria. It uses light Vibrio parahaemolyticus possesses two types of flagella. The swimmer cell energy it to move protons across the membrane. moves fast in a liquid environments, with a single polar flagellum powered by the Na + motive force,. The swarmer cell, propelled by many lateral flagella powered by H + gradient and can move slowly through highly viscous environments. 15 000 rev per second on Na ++

Sodium motive force Proton motive force

12 Proof of Bacteriorhodopsin

Photosynthesis in bacteria and light phase in chloroplasts

Photosynthesis in bacteria Bacteriochlorophyll and carotenoids (in light harvesting complexes)

No mitochondria, no chloroplasts ⇒ everything is located in the same membrane!

-e- +e - P* → P+ → P Hartmut Michel Nobel prize 1988 Structure of photosynthetic reaction centre


The light energy absorbed by an isolated pigment molecule is completely released as light and heat by process 1. In photosynthesis, by contrast, pigment undergo process 2 in the antenna complex and process 3 in the reaction center.

13 Light driven cyclic electron transfer Electron transfer in Rh. Sphaeroides Organisation of light harvesting complexes (Rh. Sphaeroides)

H+-translocation by complex III

LH2=antennae absorb light at other λ than Bchl and also provides high rate of electron delivery to RC

Light driven electron transfer Greenplants and algae Photosynthesis takes place in chromatophores Oxygen and formation

+hν ↔↔↔ ↔↔↔ • Light reactions: CO 2 + H 2O {CH 2O} + O2 {CH 2O}+ O2 CO 2 + H 2O + energy Photosynthetic organisms Animals – Need light to occur Capture of solar energy to use it for reduction Oxidation of food to obtain energy – Capture of light energy of carbon compounds – Generation of pmf and reducing power (NADPH) Animals • Dark reactions: - + ↔ O2 +4e +4H 2H 2O – Occur in light and dark – Carbohydrate synthesis Plants

14 Photosynthesis takes place in Chlorophyll Photosynthesis chloroplasts → • 6CO 2 + 6H 2O C6H12 O6 + 6 O2 • Occurs in specialised – chloroplasts • Light captured by chlorophyll – Porphyrin – Contains Mg 2+ – Green Like heme, chlorophyll a is a cyclic tetrapyrrole. One of the pyrrole rings (shown in red) is reduced. A phytol chain (green) is connected by an ester linkage. Magnesium ion binds at the center of the structure.

Photosynthesis: Light reactions Photosynthesis: Light reactions Photosynthesis: Light reactions

n H + NADP + H+ • Two light absorbing stages: Stroma Stroma – Photosystem II NADPH

– Photosystem I Q • Electron transport chains – several complexes of proteins Light Light QH 2 2e - Photosystem II • Soluble carriers: e- – Plastoquinone (Q), lipid soluble – Plastocyanin, water soluble H2O H2O Lumen + Lumen + ½O2 + 2H ½O2 + 2H n H + H+

15 Photosynthesis: Light reactions Photosynthesis: Light reactions Photosynthesis: Light reactions

NADP + Stroma H+ Stroma NADPH • Products: – Oxygen - released, essential for most on QH earth 2 Cytochrome bf Photosystem I - e complex Light - – Proton motive force - used for ATP synthesis Q e – NADPH – used in biosynthesis, the Calvin cycle

Plastocyanin Lumen Plastocyanin Lumen H+

Mitochondria related websites: Mitochondria Research Photosynthesis: Light reactions Photosynthesis: Light reactions http://www.mitochondrial.net/ Complex I home page http://www.scripps.edu/mem/ci/

• Two light absorbing stages: Joel Weiner Complex II related webpage http://www.biochem.ualberta.ca/weinerlab/FrdABCD.htm – Photosystem II The bc 1 complex home page – Photosystem I http://www.life.illinois.edu/crofts/bc-complex_site/ • Electron transport chains – several complexes of The Cytochrome Oxidase home page proteins http://www-bioc.rice.edu/~graham/CcO.html Boris Feniouk ATP synthase home page • Soluble carriers: http://www.atpsynthase.info/

– Quinone (Q), lipid soluble Antony Crofts bioenergetics course – Plastocyanin, water soluble http://www.life.illinois.edu/crofts/bioph354/ Bioenergetics course Leeds University http://www.bmb.leeds.ac.uk/illingworth/oxphos/