Respiration processes

Respiration and energy

The function of respiration is the release of energy within the cells of an organism.

This energy can be heat or chemical energy. Some of the released heat energy may be (usefully) used to maintain the temperature of the organism, but much of it will be (wastefully) lost into the environment.

The chemical energy is biologically more useful, and generally it is produced in the form of ATP - adenosine triphosphate, and the energy is effectively in the phosphate bonds. Other useful compounds are the coenzymes reduced NAD and reduced FAD. These pass on H⁺ and electrons and so can be used to reduce other compounds, or may enter a respiratory chain and thus generate more ATP.

Respiration has several stages, and these are built from a number of chemical reactions.

Several organic compounds can act as the basic fuel for these processes: the carbohydrate glucose is the main one, but there are other (classes of) substances : mostly lipids and proteins, but even ethanol (alcohol) can be respired to give energy. These are called respiratory substrates - which underlines the fact that all the chemical reactions are catalysed by/under the control of enzymes. Different substrates can enter the reactions at various stages, or be removed to take part in other reactions.

Acids and bases

Many compounds involved in respiration may be referred to as acids: pyruvic acid, lactic acid, citric acid. As they ionise and dissociate, it is nowadays more common to refer to them as composite bases (salts), e.g. pyruvate, lactate, citrate.
However in some contexts it is common practice to refer to the acid: lactic acid bacteria, citric acid cycle.
It is interesting to see how Sir Hans Krebs moves from referring to citric acid (etc) to citrate (etc) in his Nobel prize lecture.

Respiration and oxygen

Many people associate the term respiration with breathing.
Breathing - the ventilation of the respiratory surface (lungs, gills) - is best called external respiration because this identifies it as a process occuring at a distance from the main one: internal or cellular respiration, which occurs independently in all the cells of the body.

Multicellular organisms need a supply route for oxygen to individual cells, and more active animals use respiratory and blood transport systems to provide individual cells with oxygen.

Many single celled organisms (Amoeba, aerobic bacteria) and simple multicellular organisms take in (dissolved) oxygen by diffusion from their surroundings. In other words, they respire but do not breathe.

Some (simple) organisms : certain bacteria and protoctists respire without using oxygen. There are in fact several degrees of independence from oxygen:

• Obligate anaerobes need environments where there is no oxygen - they cannot grow in its presence, or are even harmed by oxygen.
  Example:Clostridium spp
• Aerotolerant bacteria do not need oxygen to grow, but can survive in its presence.
• Facultative anaerobes are capable of growing in the absence of oxygen gaining energy comes from glycolysis and fermentation. However if oxygen is present they can obtain their energy by aerobic respiration.
  Example: Escherichia coli

Glycolysis

Glycolysis is the first stage of respiration. In the cell it takes place in the cytoplasm (also known as cytosol), i.e. not inside mitochondria. It does not require oxygen, so it may be described as anaerobic.

Glycolysis means splitting of glucose, a 6-carbon sugar, to produce pyruvate, a 3-carbon compound. The main reaction stages are as follows:


There is a net gain of 2 ATPs and 2 reduced NADs in the process. No CO₂ is given off.

Subsequent stages can take quite different directions, but there are not different versions of glycolysis.

Glycolysis stages - inside the cell

The section enclosed in blue boxes takes place twice, as 2 molecules of triose phosphate [3C] are obtained from one glucose molecule [6C] at the start.

Glycolysis in more detail

Glycolysis takes place in a series of 10 reactions. Each is controlled by its own enzyme, and the product of each takes part in the next reaction.

[1] The first reaction involves activating glucose with the input of energy in the form of ATP - a phosphorylation reaction. This produces glucose (6-)phosphate, and the enzyme involved is called hexokinase or glucokinase (kinase implies getting things moving). This has the effect of stimulating the intake of more glucose into the cell via glucose transporters (GLUTs) which are proteins embedded in cell membranes.

This part is bypassed when glycogen is being broken down in muscles and liver: glycogen phosphorylase phosphorylates glucose residues as it releases them from the tips of polyglucose branches in the glycogen molecule.

[2] This G6P is converted into fructose 6-phosphate (F6P), (another 6-carbon, 1 phosphate sugar), by the enzyme glucose phosphate isomerase (G6P and F6P are isomers).

[3] F6P is converted into F1,6BP (fructose 1,6 bis-phosphate - meaning fructose with 2 separate phosphate groups, at different ends of the molecule). The enzyme involved is phosphofructokinase (PFK), and the second phosphate group also comes from ATP, which then becomes ADP. Incidentally, ADP has 2 phosphate groups and ATP has 3, attached to the same part of the molecule.

[4] Then F1,6BP (6-carbon, 2 phosphate sugar) is broken down, using the enzyme aldolase, into two molecules of triose phosphate TP (3-carbon, 1 phosphate sugar). In fact these are slightly different: one is glyceraldehyde 3-phosphate, and the other is dihydroxyacetone phosphate DHAP - isomers of one another.

[5] Triose phosphate isomerase converts dihydroxyacetone phosphate into glyceraldehyde 3-phosphate so each of the next stages take place twice.

[6] The two triose phosphates then each accept another phosphate group - this time from inorganic phosphate Pi - and become oxidised, under the action of the enzyme triose phosphate dehydrogenase. This forms 1,3-bisphosphoglycerate: G 1,3 BP (conjugate base of 1,3-bisphosphoglyceric acid: 3-carbon, 2 phosphate).


[7] G 1,3 BP gives up one phosphate group, producing G3P and releasing (2x) ATP as a result.

[8,9] G3P is converted into other 3 carbon, 1 phosphate compounds; G2P, PEP.

[10] Under the influence of pyruvate kinase, PEP gives up the last phosphate group, producing pyruvate, a 3 carbon compound, and releasing (2x) ATP as a result.

Anaerobic respiration

In the absence of oxygen (anoxic conditions), pyruvate cannot be completely broken down.

The NAD used up in glycolysis needs to be replaced, and the next stages use reduced NAD, so that NAD is regenerated, allowing glycolysis to continue.

There are two alternatives, depending on the type of organism involved.

In animal cells:
Pyruvate → lactate (another 3-carbon compound)

In plant cells (including yeast):
Pyruvate → ethanol (a 2-carbon compound) + carbon dioxide (a 1-carbon compound)

Why is it an advantage for animals to perform the first reaction, rather than the second one?
>If the combined efforts of the respiratory system and circulatory system cannot provide enough oxygen in a short time frame, then they could not get rid of carbon dioxide at the same time.

What functions are performed by a fermentation lock?

>Allows CO₂ gas to escape (no buildup of pressure)

>Prevents entry of oxygen (keeps it anaerobic)

>Prevents entry of contaminants (bacteria/other microbes, fruit flies(??), etc)
>Wine could otherwise turn to vinegar/ ethanoic (acetic) acid - spoiling the wine!

Consequences of anaerobic respiration

The main products of anaerobic respiration are inhibitory.

In animals that normally respire aerobically, 'switching to' anaerobic respiration in the absence of oxygen allows activity to continue at a limited extent for a brief time. A likely scenario might include running to escape from a predator, or human exercise. In both these cases, the supply of oxygen breathed in is exceded by the oxygen requirements of muscles involved.

Lactate is the composite base of lactic acid, which is often thought to cause muscle fatigue and exhaustion, although this is not simply a pH effect. The build-up of lactate/lactic acid eventually causes muscular activity to reduce. After activity, it is subsequently removed from muscles and sent via the bloodstream to the liver, where it is gradually converted back to pyruvate under aerobic conditions, and this can be re-converted to glucose via gluconeogenesis. Athletic training habituates the body to this buildup.

The cornea - the front surface of the eye - is one of the few parts of the body that receives oxygen directly from the air, rather than from blood. Wearers of hard contact lenses used to have to gradually build up their wearing time, allowing cells to develop tolerance to lactic acid produced as a result of anaerobic respiration, but modern hard lenses are now much more "gas permeable". Soft lenses are inherently permeable, so this is not a problem.

Lactobacillus spp produce lactic acid as a result of fermentation of lactose in cheese and yoghurt production, and when milk goes sour. Lactobacilli are also involved in the production of soy sauce.

Ethanol is also rather toxic. Yeast respires aerobically but if air supply is reduced it can also perform anaerobic respiration - also called alcoholic fermentation. The maximum amount of ethanol (alcohol) tolerated depends on the type of yeast - usually 10-15%.

Roots of plants normally respire aerobically, but in waterlogged conditions oxygen supply is reduced and certain plants can tolerate more ethanol than others. An example is rice which normally grows in paddy fields which are routinely flooded.

Aerobic respiration

Aerobic respiration involves mitochondria. Pyruvate from glycolysis is taken inside by 'active transport'. It passes through the two membranes and enters the inner matrix of the mitochondrion. Since one glucose molecule produces 2 pyruvate molecules, all the resulting compounds need to be 'doubled up'.

The link reaction

Pyruvate is oxidised to 'acetate', losing carbon dioxide and producing reduced NAD in the process.
The carbon dioxide diffuses out of the mitochondrion.

The carrier molecule coenzyme A combines with 'acetate', forming acetylcoenzyme A.
This is effectively a 2-carbon compound (attached to a much larger molecule).

The link reaction in a mitochondrion

Ins and Outs of mitochondria

The outer mitochondrial membrane has protein channels called porins which allow certain small molecules to pass by facilitated diffusion into and out of the intermembrane space.

Mitochondria need to take in (via the inner membrane): pyruvate, oxygen, ADP + inorganic phosphate, and to release CO₂ and ATP.

Pyruvate from glycolysis is taken into the mitochondrion in co-transport with a proton (H⁺) by a transporter protein pyruvate translocase, located in the inner mitochondrial membrane. This is a secondary active transport process, the protons having been deposited in the intermembrane space by the electron transport chain (see later).
Pyruvate thus enters the inner matrix of the mitochondrion . . .

The link reaction in more detail

The enzyme pyruvate dehydrogenase complex has 3 sections, each containing large numbers of copies (30, 60, 12) of three enzymes:
The first decarboxylates pyruvate using the coenzyme TPP (thiamine pyrophosphate), a derivative of vitamin B1, releasing CO₂.
The second transfers the resulting acetyl group (-CH₃C=O) to coenzyme A, releasing acetyl coenzyme A.
The third performs a dehydrogenation reaction on an intermediate lipoamide compound using FAD as a permanently attached prosthetic group. The resulting reduced FAD passes on hydrogen to NAD, so reduced NAD is released.

Carbon dioxide diffuses out of the mitochondrion, acetyl coenzyme A enters the Krebs cycle in the mitochondial matrix, and reduced NAD interacts with the electron transport chain in the inner membrane wall.

The Krebs cycle

The Krebs cycle is a series of 9 oxidation-reduction reactions, and molecular reorganisations.

Acetylcoenzyme A reacts with a four-carbon molecule, releasing coenzyme A and producing citrate, a six-carbon molecule. The coenzyme A returns to pick up more pyruvate.

At 4 points, dehydrogenation reactions (oxidation) occur.
NAD and FAD are coenzymes that take part in these processes, acting as hydrogen acceptors. As a result 3 molecules of reduced NAD and 1 molecule of reduced FAD are produced.

At one stage enough energy is released to result in the production of ATP by substrate-level phosphorylation:
ADP + Pi [+energy} → ATP

At two stages carbon dioxide is lost, so that eventually the four-carbon compound is re-formed.
This is then ready to react with Acetylcoenzyme A again ...

Other respiratory substrates can enter the Krebs cycle

Other equivalent chemical compounds such as lipid breakdown products, and deaminated amino acids, can enter the cycle and be converted into the next compound, with the release of energy.

Fatty acids are an extremely efficient source of energy, as they consist of a chain of (usually 16 or 18) carbon atoms, [with associated H atoms]. They can be taken into mitochondria via special carrier proteins. These then undergo a breakdown process involving removal of pairs of carbon atoms, which are converted to acetylcoenzyme A. This is then processed by the Krebs cycle in exactly the same way as products of carbohydrate origin, providing ATP as a result of aerobic respiration.

Under more challenging conditions, protein can be used as a respiratory substrate. Amino acids are deaminated, and the resulting keto acids can enter aerobic respiration at a number of points. Deamination products of several amino acids can be converted into pyruvic acid or acetylcoenzyme A, and others enter the Krebs cycle at various points.

One cycle, three names

The Krebs cycle is also known as the citric acid cycle or the tricarboxylic acid cycle.
Each stage is controlled by its own enzyme within the mitochondrial matrix. Once again, products are displayed in bold.
Coenzyme A also contributes as reactant in this cycle (step 5 below) - once again causing decarboxylation (removal of carbon as carbon dioxide).
At 3 stages, water enters the cycle as a reactant.

[1] Under the influence of the enzyme citrate synthase, acetyl coenzyme A combines with oxaloacetate to form citrate, (releasing coenzyme A, enabling it to return to the pyruvate dehydrogenase complex and pick up more acetyl groups, or succinyl groups - see [5] below).
Acetyl is effectively a 2-carbon compound, and oxaloacetate has 4 carbons, so citrate is a 6-carbon compound.

[2,3] Aconitase catalyses the conversion from citrate to cis-aconitate, then isocitrate.

These are all (6-carbon) tricarboxylic acids.

[4] Isocitrate is oxidised by isocitrate dehydrogenase forming alpha ketoglutarate with the production of reduced NAD, and CO₂ is given off. The product is a 5-carbon compound.

[5] Alpha ketoglutarate is oxidised by alpha ketoglutarate dehydrogenase with the input of coenzyme A, forming succinyl coenzyme A, also resulting in the production of reduced NAD, and CO₂ is given off. The product is a 4-carbon compound, and all the following compounds are also 4-carbon compounds.

[6] Under the influence of succinyl-CoA synthetase, succinyl coenzyme A forms succinate and releases coenzyme A, producing GTP then ATP by substrate level phosphorylation

[7] Succinate is oxidised by succinate dehydrogenase to fumarate, producing reduced FAD. Succinate dehydrogenase is part of complex II of the electron transport chain and is attached to the inside of the mitochondrial membrane.

[8] Fumarate is rearranged by fumarase into malate

[9] Malate is oxidised by malate dehydrogenase, producing reducd NAD and oxaloacetate, which allows the cycle to continue ...

Electron transfer chain

Reduced NAD and reduced FAD provide electrons and protons (H⁺).

Electrons are transferred along a chain of molecules which participate in oxidation-reduction reactions, effectively losing energy at each stage.

Protons are passed across the inner mitochondrial membrane, using the energy of the electrons. They build up in the space between the mitochondrial membranes, and their concentration gives a source of energy for the final (ATP production) process.

Oxygen acts as the final electron (and proton) acceptor in the transfer process, and water is produced.

Protons pass back from the intermembrane space through the enzyme ATP synthase, which results in the production of ATP:
ADP + Pi [+energy] → ATP

As a result of this process (oxidative phosphorylation), several molecules of ATP are produced in addition to those formed by substrate level phosphorylation in glycolysis and the Krebs cycle.

Et Cetera Et Cetera

There are 4 enzyme complexes fixed in position in the inner membrane of the mitochondrion, and other compounds which move within or across the surface of the phospholipid bilayer, acting as shuttles moving electrons and protons (hydrogen ions, H⁺) on to the next enzyme complex.

Reduced NAD and reduced FAD have different entry points, as they have different energy levels (reduced FAD is lower). They both provide electrons which give energy to the enzyme complex, and are then passed to the next in the series. At each stage the electrons have less energy.

Three of the 4 enzyme complexes take protons and deposit them on the other side of the inner mitochondrial membrane, in the intermembrane space, using energy from the electrons. This could be called pumping of protons, but not active transport, which uses ATP. These protons accumulate so they are eventually at a higher concentration than in the mitochondrial matrix.

At the end of the chain, electrons and protons finally combine with oxygen to form water
4 H⁺ + 4 e^- + O₂ → 2 H₂O.

Protons pass back from the intermembrane space to the matrix via the enzyme ATP synthase. For every 3 protons that move down the tubular section and out via the rotating end section, one ATP molecule is formed (from ADP + Pi). Because of the movement of protons across the membrane down a concentration gradient, it is called chemiosmotic, a reference to the movement of water molecules across membranes by osmosis.

Although the last two stages are slightly separate, this process is called oxidative phosphorylation.

Balancing the equation

Anaerobic respiration

In animal cells:
C₆H₁₂O₆→ 2 C₃H₆O₃
Glucose → lactic acid
"molecular reorganisation" - no CO₂ or H₂O given off
In plant cells (including yeast):
C₆H₁₂O₆→ 2 C₂H₅OH + 2 CO₂
Glucose → ethanol + carbon dioxide

Aerobic respiration

The whole reaction :
C₆H₁₂O₆ + 6 O₂ → 6 CO₂ + 6 H₂O
glucose + oxygen → carbon dioxide + water
The carbon dioxide is produced in the mitochondrial stages (2x CO₂ molecules from the link reaction, 4x CO₂ molecules from the Krebs cycle), but neither of these take in oxygen directly.

The water comes from the electron transport chain.
But 6 molecules of oxygen result in 12 molecules of water: 6 O₂ + 24 H⁺ + 24 e^- →12 H₂O.
The other 6 molecules of water are actually taken in as reactants in the Krebs cycle.

Accounting for protons, and energy

Glycolysis - net gain: 2 ATP, 2 reduced NAD - but these need to be transported from the cytosol into the mitochondrial matrix. There are 2 possible mechanisms for this: the malate-aspartate shuttle or the G3P shuttle, which effectively provides a reduced FAD to the electron transport chain, and so produces less ATP.

Link reaction: 2 reduced NAD (delivered to the electon transport chain ETC)

Krebs cycle: 2 ATP, 3x2 reduced NAD (delivered to the ETC), 1x2 reduced FAD (to ETC)

ETC
Each reduced NAD pumps 10 protons (4+4+2 from complex I, III and IV), each reduced FAD produces 6 protons (4+2 from complex III and IV).
- Some debate about the basis for the outcome in terms of ATP molecules produced:
Conventional calculation:
Each reduced NAD produces 3 ATPs, each reduced FAD produces 2 ATPs.
More recent estimates:
Each reduced NAD produces 2.5 ATPs, each reduced FAD produces 1.5 ATPs.
This lower figure may be based on the transport of ATP and ADP (see below).

ATP synthesis
The beta subunits at the base of ATP synthase directly cycle through 3 states in the the production of ATP.
It now seems wrong to say that 3 protons are used in the production of one molecule of ATP, and it depends more on the geometry of the c-ring above this.
A more accurate figure for Metazoa (many-celled animals) is 2.7 H⁺ per ATP, but it varies in other species (5 in some Eubacteria).

Other uses for protons
Protons are also lost from the intermembrane space as ATP is exported from the mitochondria into the cytosol together with the movement of ADP (and phosphate?) in the other direction using ATP-ADP translocase which operates as an antiporter, by co-transport with H⁺. Pyruvate translocase (see above) also uses a similar system for the entry of pyruvate into the mitochondria.

The bottom line

Anaerobic respiration: only 2 ATP, from glycolysis (reduced NAD must be recycled)

Aerobic respiration (based on conventional values): 38 ATP
(2 from glycolysis, 2 from Krebs, 30 from NAD, 4 from FAD)

Aerobic respiration (based on more recent values): 32 ATP
(2 from glycolysis, 2 from Krebs, 25 from NAD, 3 from FAD)
OR
Aerobic respiration (based on more recent values and G3P shuttle for reduced NAD/FAD from glycolysis): 30 ATP
(2 from glycolysis, 2 from Krebs, 22 from NAD, 4 from FAD)

So aerobic respiration is 19, 16 or 15 times more efficient than anaerobic respiration.