22 1. 2 2 x C 3 2 x C 2 Cytosol Glucose pyruvate 2 x CO 2 4 x CO 2 glycolysis 1 x C 6 Mitochondrion...

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2

3

2 x C3

2 x C2

Cytosol Glucose pyruvate

2 x CO2

4 x CO2

glycolysis

1 x C6

Mitochondrionpyruvate 3 CO2CAC

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The Citric Acid Cycle

A series of 8 reactions in the mitochondrial matrix.

oxidizes the C2 acetyl component of acetyl-CoA to 2 molecules of CO2

& energy in the form of reduced coenzymes, 3NADH and FADH2.

The cycle carries his name, the Krebs cycle.Because the 1st intermediate in the cycle is citric acid, it is also referred to as the Citric Acid Cycle.Because citric acid is a tricarboxylic acid, it is also referred to as TCA cycle.

Hans Adolf Krebs (1900-1981)

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At the pyruvate position:The enzyme that initiates it, pyruvate dehydrogenase(PDH) is inhibited by ATP and NADH because these are abundant when energy is readily available. It is activated by ADP, which is abundant when the cell needs energy.

At the CAC position: Rxn #3 - isocitrate dehydrogenaseActivated by ADP and NAD+

inhibited by ATP & NADH

Rxn #4 – ketoglutarate dehydrogenase ADP activates

NADH & succinyl-CoA inhibits CAC

Regulation of CAC

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One turn of CAC

acetyl CoA + 2 H2O + 3 NAD+ + FAD + GDP + Pi 3 NADH + 3H+ + FADH2 + GTP + HS-CoA + 2 CO2

The main function of CAC is to oxidize pyruvate and reduce NAD+ and FAD in order to produce high E compounds (NADH and FADH2) required for ATP synthesis.

With pyruvate

Pyruvate + HS-CoA + NAD+ acetyl CoA + NADH + H+ + CO2

Overall

Pyruvate + 4 NAD+ + FAD + GDP + Pi + 2 H2O 4 NADH + 4H+ + FADH2 + GTP + 3 CO2

If we take into account glycolysis and the pyruvate dehydrogenase reaction, and the fact that one molecule of glucose generates two molecules of pyruvate, we can write an equation for the catabolism of glucose through glycolysis and the CAC as:

Glucose + 2 H2O + 10 NAD+ + 2 FAD + 4 ADP + 4 Pi 10 NADH + 10 H+ + 2 FADH2 +

4 ATP + 6 CO2

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Most of the ATP generated during glucose oxidation is not formed directly from reactions of glycolysis and CAC, but rather from the reoxidation of reduced electron carriers in the respiratory chain.

These electron carriers, NADH and FADH2, have a high redox potential, in the sense that

their oxidation is highly exergonic.As electrons are transferred from the reduced carriers to molecular oxygen, in a stepwise fashion, the energy released is used to drive the synthesis of ATP from ADP, producing about

2.5 moles of ATP per mole of NADH and 1.5 moles of ATP per mole of FADH2.

NADH is a stronger reducing agent than FADH2 and therefore can produce ~1 more ATP.

NADH and FADH2 are reoxidized by electron

transport proteins bound to the inner mitochondrial membrane.

A series of linked oxidation reduction reactions occurs, with electrons being passed along a series of electron carriers known as the electron transport chain, or respiratory chain.

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The reduced coenzymes formed in the citric acid cycle enter the electron transport chain and the electrons they carry are transferred from one molecule to another by a series of oxidation–reduction reactions. Each reaction releases energy until electrons and protons react with oxygen to form water. 

Electron Transport

2 H+ +2 e- + ½ O2 H2O

The energy released during electron transport is used to synthesize ATP from ADP and Pi in a process called oxidative phosphorylation.

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The electron acceptors in this transport system are called electron carriers.A molecule with a high reduction potential, , tends to be reduced if paired with a molecule with a lower reduction potential.

There are four types of electron carriers:

Flavins: FMN FMNH2

oxidized reduced

Iron-sulfur : Fe3+ + e- Fe2+

clusters oxidized reduced

Coenzyme Q; Q QH2

oxidized reduced

Cytochromes: Fe3+ + e- Fe2+

oxidized reduced

Flavin mononucleotide

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Coenzyme Q

Fe-S cluster = nonheme iron protein

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The different types of cytochromes are designated a lowercase letter (a, b, c); further distinctions are done with subscripts, as in C1.

Cytochromes identified as: a, a3, b, c, c1

Cytochromes

proteins that contain heme groups in which the iron cycles between Fe+2 and Fe+3 and proteins with iron–sulfur groups in which the iron also cycles between Fe+2 and Fe+3 .

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Electron transport is a sequence of reactions that facilitate the flow of electrons from

NADH and FADH2 all the way to the reduction of oxygen to form water:

The membrane has four distinct protein complexes: complex I, II, III, IV.Each complex is a multienzyme system and contain electron carriers needed for electron transport.

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Complex IV – Cytochrome c oxidase. Contains about 10 subunitsHere electrons are transferred from cytochrome c to cytochrome a, and then to cytochrome a3, the last cytochrome.In the final step of electron transport, electrons and H+ combine with oxygen to form water:

2 H+ + 2 e- + ½ O2 H2O

Complex I and II receive electrons from the oxidation of NADH and FADH2, respectively, and pass them

along to a lipid soluble electron carrier, coenzyme Q, which moves freely in the membrane.

Complex III catalyzes the transfer of electrons from the reduced form of coenzyme Q to cytochrome c, a soluble protein electron carrier that is mobile within the intermembrane space.

Complex IV catalyzes the oxidation of cytochrome c, reducing O2 to water. The energy released by these exergonic reactions is used to pump protons from the matrix into the inner membrane space, creating a proton gradient across the inner membrane.

Complex I, III, and IV create a proton gradient across the inner mitochondrial membrane, and the energy of this gradient is utilized by complex V to synthesize ATP from ADP.

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Oxidative Phosphorylation

The energy-releasing oxidation reactions give rise to a pH gradient across the inter mitochondrial membrane.

For a pair of electrons entering the respiratory chain as NADH and traversing the entire chain to O2, the energy change is about 218 kJ/mol (69.5+36.7+112):

NADH + H+ + ½ O2 NAD+ + H2O ΔG˚̛. = -218 kJ/mol

ΔG for ATP hydrolysis under intracellular conditions is estimated to be -50 kJ/mol, so the synthesis of 2.5 mol ATP requires at least 2.5 x 50 = 125 kJ. Therefore, enough E is available to drive ATP synthesis.

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The process where the energy generated through the electron transport is coupled with the

synthesis of ATP is called oxidative phosphorylation.

This pH gradient generates an electrochemical potential difference, a voltage drop, across the membrane. The energy possessed by the voltage difference is coupled to the synthesis of ATP.

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Chemiosmotic coupling

To explain how the H+ gradient is used by complex V to synthesize ATP, Peter Mitchell proposed the chemiosmotic model in 1961.

“The free energy from electron transport drives an active transport system (transport against a conc. gradient), which pumps protons out of the matrix into the intermembrane space. This action generates an electrochemical gradient for protons. The protons on the outside have a thermodynamic tendency to flow back in, down their electrochemical gradient, and this flow provides the energy for ATP synthesis”.

To equalize the H+ concentrations, these use a protein complex called ATP synthase, which allows them to return to the matrix.

As the protons flow, energy is generated and used to drive ATP synthesis:ADP + Pi + energy ATP

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Therefore, proton pumping results in the conversion of the energy of the electron transport to osmotic energy in the form of an electrochemical gradient. This is a concentration gradient that also establishes an electrical potential.The energy released from discharging this gradient can be coupled with phosphorylation of ADP to ATP by means of complex V, ATP synthase.

The proton gradient leads to ATP production by means of ion channels present in ATP synthase.

The F0 part of the protein is the proton channel and permits the passage of H+

The F1 part of the protein catalyzes the phosphorylation reaction

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Conformational couplingThe effect of the proton flux is to cause a conformational change that leads to the release of already formed ATP from ATP synthase.

Protons flow through F0 generating energy and turning . Just as the flow of water turns a water wheel. This allows for the active sites to turn shape:

F1 consists of a central subunit and three protein subunits with 3 active sites: loose (L)tight (T)open (O)These states interconvert as a result of the proton flux through the synthase.

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ADP + Pi enter (O)It converts into (L) and ADP & Pi react Then (L) turns into (T) which has little affinity for ATP and releases it.

The activity of electron transport & oxidative phosphorylation depends on ADP and ATP levels.↑ ADP activates ↓ ATP deactivates

In essence, the chemical energy of the proton gradient is converted to mechanical energy in the form of rotating proteins. This mechanical energy is then converted to the chemical energy stored in the high-energy phosphate bond of ATP.

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Cytoplasmic NADH is not permeable to the mitochondria. In order to extract the energy from this NADH, the reducing equivalents must be transferred into the mitochondrion. Therefore, the reducing equivalents must be shuttled to the inner mitochondrial membrane without physical movement of NADH.

This process involves the reduction of a substrate by NADH in the cytoplasm, passage of the reduced substrate into the mitochondrial matrix, and passage of the oxidized substrate back to the cytoplasm, where it can undergo the same cycle again.

There are two shuttle systems:

1. Dihydroxyacetone phosphate/glycerol-3-phosphate shuttle is active in brain and skeletal muscle.

2. Malate/aspartate shuttle is active in liver, kidney and heart.

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1. Dihydroxyacetone phosphate/glycerol-3-phosphate

This process involves the reduction of FAD by G3P followed by transfer of an electron pair from FADH2 to coenzyme Q, just like intramitochondrial NADH transfers electrons to CoQ.

G/P shuttle NADH (cytosol) FADH2 (mitochondrion)

2 ATP

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2. Malate/aspartate

This process involves the reduction of NAD+ by malate followed by transfer of an electron pair from NADH to complex I, just like intramitochondrial NADH transfers electrons to complex I.

M/A shuttle NADH (cytosol) NADH (mitochondrion)

3 ATP

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Considers NADH from glycolysis using the M/A shuttleWhere 2 NADH 2 FADH2

Based on your textbook

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