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How is Coenzyme A Transported to the Matrix?

How is Coenzyme A Transported to the Matrix?


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So, I've been researching cellular respiration on my own, and trying to keep track of most of the major processes. However, I do have one question left: I can't seem to find any sort of information about how Coenzyme A (I think it's made in the cytosol) is transferred to the matrix (the inside of the mitochondria, inside the inner membrane). Is it transported with a shuttle like for NAD+/NADH, a translocase like for ATP/ADP, or something else? Thank you for your time.


Zhyvoloup et al. (2003) J Biol Chem 278: 50316-50321; emphasis mine:

CoA synthase is localized on the mitochondrial outer membrane. Moreover, we demonstrate for the first time that phosphatidylcholine and phosphatidylethanolamine, which are the main components of the mitochondrial outer membrane, are potent activators of both enzymatic activities of CoA synthase in vitro. Taken together, these data provide the evidence that the final stages of CoA biosynthesis take place on mitochondria and the activity of CoA synthase is regulated by phospholipids.


https://www.ncbi.nlm.nih.gov/pubmed/14514684


I think this can clear your doubt… As it shows how the transport is done between the matrix and cytosol… And it also explains the presence of coenzyme A ie CoA SH at both the sites.


How is Coenzyme A Transported to the Matrix? - Biology

Ubiquinol/Ubiquinone or Coenzyme Q10

Coenzyme Q10 is a redox active lipid-soluble compound which is synthesized by animals, plants and bacteria. Coenzyme Q10 is present in most cellular membranes. The major form of coenzyme Q10 in human and in most of mammals has 10 isoprene units in the side chain. Such polyisoprenoid side-chain is responsable for anchoring to the molecule to lipid-rich structures. Q10 is an essential carrier for the electron transfer in the mitochondrial respiratory chain for ATP production, and also it acts as an important antioxidant in the body. [ 1, 2, 3, 4 ]. Q10 exists in the oxidized (UQ, ubiquinone), partially reduced (ubisemiquinone radical, UQ .- ) and in the reduced form (ubiquinol, UQH2). Coenzyme Q10 undergo reversible redox cycling between the three states. Such redox cycling define coenzyme Q10 its function as electron carrier in the mitochondrial respiratory chain. Coenzyme Q10 transfers electrons from complexes I and II to complex III [ 1, 5, 6 ]. The reduced form (ubiquinol) is a major form of coenzyme Q10 in the cell. Coenzyme Q10 is acting through formation of its semiquinone radical is a major source of cellular and mitochondrial superoxide radical [ 5, 6, 7 ].

Coenzyme Q10. Ubiquinol/Ubiquinone and semiquinone radical [ 8, ].


Coenzyme Q10 has very low toxisity is used as dietary supplement for more than 30 years. Rre-clinical and clinical trails shown that coenzyme Q10 does not cause serious adverse effects in humans. Futhermore, studies on animals and humans suggest that dietary coenzyme Q10 supplementation does not influence biosynthesis of coenzyme Q10 in the cell and does not resulting in accumulation in plasma or tissues. The maximum tolerated doses of coenzyme Q10 were estimated to be in range 250 mg/kg - >4,000 mg/kg for mice and rats. The lethal dose for coenzyme Q10 was greater than 5000 mg/kg for male and female rats [ 9, 10, 11 ].

Respiratory chain electrons flow from NADH to flavin mononucleotide in complex I and are then transferred to the set of Fe-S clusters. The terminal Fe-S cluster interacts with ubisemiquinone radical and, therefore, is thought to be the electron donor to ubiqinone. Transfer of the first electron results in the transient formation of ubisemiquinone radical, and transfer of the second electron reduces the ubisemiquinone radical to the fully reduced UQH2. In this reaction ubisemiquinone radical can react with oxygen to form superoxide [ 1, 12, 13 ]. Reverse electron transport is based electron flow from succinate through complex II to ubiqinone and then to complex I, which finally reduces matrix NAD + [ 1, 13, 14 ]. Q-cycle is a chain of processes where electrons flow from UQH2 to cytochrome c at complex III. UQH2 binds to the Q(o) site and transfers the first electron to the Rieske iron-sulfur protein (RISP) forming unstable ubisemiquinone radical. This radical donates the second electron to the low-potential heme (bL) of cytochrome b and is then conveyed to the high-potential heme (bH) near the 'in' side (the matrix side) of the membrane. Electron from bH passes to a ubiqinone at the second ubiqinone-binding site, Q(i) resulting in formation of a stable ubisemiquinone radical. At second part of Q-cycle one additional UQH2 molecule is oxidized as described above. Final result of Q cycle is oxidation of two UQH2 and generation of one UQH2 at the Q(i) site, reduction of two cytochrome c molecules, and depositing of four protons into the intermembrane space [ 1, 15 ].

Functions of Ubiquinone (UQ) in the Mitochondrial Respiratory Chain (RC). In normal forward electron transfer, UQ accepts electrons from complexes I and II and passes them singly to complex III. At complex III, the 'Q cycle', which allows pumping of protons from the matrix into the intermembrane space, involves two distinct UQ-binding sites. UQH2 is reduced at the Q(o) site, passing one electron to cytochrome c (cyt c) and the other down to the Qi site, where the electron is given to a bound UQ during the first cycle, forming UQ .- , or to a bound UQ .- generated during the first cycle. Oxidized UQ formed at the Q(o) site and UQH2 formed at the Q(i) site after completion of the 'Q cycle' are free to diffuse out into the UQ pool. As electrons are transported, they may leak to oxygen, forming superoxide (O2 .- ). Red stars indicate potential sources of O2 .- production. Superoxide dismutase (SOD) converts O2 .- to hydrogen peroxide (H2O2) that is reduced to water by glutathione peroxidase (GPX). Both O2 .- and H2O2 have been implied in modulating the function of signal transduction pathways ('other reactions' in the figure). UQ also accepts electrons from several non-RC dehydrogenases, including the mitochondrial glycerol-3-phosphate dehydrogenase (G3PDH), dihydroorotate dehydrogenase (DHODH), and electron transfer flavoprotein oxidoreductase (ETFQOQ) (see main text for other dehydrogenases not shown in the figure). Uphill electron transfer from UQH2 to NAD + through complex I is known as reverse electron transport. The I-III-IV supercomplex, which is the most active supramolecular form, is schematically shown on the left of the figure. [ 1, ].

The reduced form of coenzyme Q10 (UQH2) is an effective antioxidant and and inhybitor od lipid peroxidation. Antioxidant properties of coenzyme Q10 are based on reversible redox cycling between the three states:

The primary role of coenzyme Q10 is the prevention of lipid peroxyl radicals (LOO .- ) production during initiation. UQH2 reduces the initiating perferryl radical with the formation of ubisemiquinone radical and H2O2. Direct reaction of UQH2 eliminates LOO .- is possible [ 16, 17, 18, 19 ].

Sites of action of ubiquinone on lipid peroxidation. LH - polyunsaturated fatty acid OH . - hydroxyl radical Fe 3+ -O2 .- - perferryl radical CoQH2 - reduced coenzyme Q CoQH .- - ubisemiquinone radical, L . - carbon-centered radical LOO . - lipid peroxyl radical LOOH - lipid hydroperoxide VitE . - α-tocopheroxyl radical Asc . - ascorbyl radical [ 16, ].

In addition, the role of coenzyme Q10 in defending proteins from oxidation has been proposed [ 20 ].

Cytosolic NADPH-dependent reductase, lipoamide dehydrogenase DT-diaphorase, glutathione reductase and thioredoxin reductase (TrxR1) are able to reduce oxidized form of coenzyme Q10. Thioredoxin reductase is shown as most effective reductant of coenzyme Q10 [ 21, 22, 23, 24 ].

Coenzyme Q10 biosynthesis is complex process and current knowledge derives mainly from the characterization of coenzyme Q10 intermediates in coenzyme Q10 defficient bacterial and yeast mutant strains. Yeast Saccharomyces cerevisiae coenzyme Q10 biosynthesis involve the products of at least nine genes designated Coq1 to Coq9 [ 25, 26, 27, 28, 29 ].

Schematic illustration of the ubiquinone (UQ) biosynthetic pathway in the yeast Saccharomyces cerevisiae. Ubiquinone biosynthesi in Saccharomyces cerevisiae pathway starts with assembly and elongation of the isoprenoid tail catalyzed by the enzyme Coq1p. Coq2p mediates the condensation of the isoprenoid tail with either one of two basic ring structures, para-hydroxybenzoate (4-HB) or para-aminobenzoate (pABA), producing 3-hexaprenyl-4-hydroxybenzoate (HHB) and 3-hexaprenyl-4-aminobenzoic acid (HAB) respectively. The basic ring moiety then undergoes a series of modifications to produce UQ. NH2-to-OH conversion is thought to takes place prior to demethoxyubiquinone (DMQ6) formation. Enzymes required for 5 of the 7 modifications are shown in blue. A question mark (?) indicates that the protein catalyzing the reaction has yet to be identified. The intermediates that have been detected in yeast coq mutants are shown in brackets. Asterisks indicate compounds that are the main intermediates detected when either 4-HB or pABA are provided as ring precursors. Coq8p is a putative kinase, believed to have a regulatory role in UQ6 biosynthesis. NH2 from pABA and C4-aminated intermediates are shown in red. [ 27, ].

Ubiquinone biosynthesis is highly conserved among prokaryotes. Biosynthesis of E. coli Coenzyme Q8 requires nine ubi genes. Most of them are encoding enzymes that modifying the aromatic ring of the 4-hydroxybenzoate universal precursor [ 25, 26, 30, 31 ].

Biosynthetic pathway of ubiquinone in Escherichia coli. The numbering of the aromatic carbon atoms is shown on coenzyme Q8, and the octaprenyl tail is represented by R on C-3 of the different biosynthetic intermediates. The name of the enzymes catalyzing the reactions (each labeled with a lowercase letter) is indicated. Abbreviations used for 4-hydroxybenzoate (4-HB), 3-octaprenyl-4-hydroxybenzoate (OHB), 3-octaprenylphenol (OPP), coenzyme Q8 (Q8), C1-demethyl-C6-demethoxy-Q8 (DDMQ8), and C6-demethoxy-Q8 (DMQ8) are underlined. The XanB2 protein, present in some prokaryotes but not in E. coli, catalyzes the production of 4-HB from chorismate. [ 31, ].


Formation of Acetyl-CoA through the Transition Reaction

The transition reaction connects glycolysis to the citric acid (Krebs) cycle. Through a process called oxidative decarboxylation, the transition reaction converts the two molecules of the 3-carbon pyruvate from glycolysis (and other pathways) into two molecules of the 2-carbon molecule acetyl Coenzyme A (acetyl-CoA) and 2 molecules of carbon dioxide. First, a carboxyl group of each pyruvate is removed as carbon dioxide and then the remaining acetyl group combines with coenzyme A (CoA) to form acetyl-CoA.

Figure (PageIndex<1>): The Transition Reaction between Glycolysis and the Citric Acid Cycle. Before the pyruvates from glycolysis can enter the citric acid cycle, they must undergo a transition reaction. The 3-carbon pyruvate is converted into a 2-carbon acetyl group with a carboxyl being removed as CO 2 . The acetyl group is attached to coenzyme A to form acetyl coenzyme A (acetyl-CoA), a key precursor metabolite. As the two acetyl groups become oxidized to acetyl-CoA, two molecules of NAD + are reduced to 2NADH + 2H + .

As the two pyruvates undergo oxidative decarboxylation, two molecules of NAD + become reduced to 2NADH + 2H + (Figures (PageIndex<1>) and (PageIndex<2>)). The 2NADH + 2H + carry protons and electrons to the electron transport chain to generate additional ATP by oxidative phosphorylation.

Figure (PageIndex<2>): The Transition Reaction between Glycolysis and the Citric Acid Cycle

The two molecules of acetyl-CoA then enter the citric acid cycle. The 2NADH molecules that are produced carry electrons to the electron transport system for further production of ATPs by oxidative phosphorylation.

The overall reaction for the transition reaction is:

2 pyruvate + 2 NAD + + 2 coenzyme A

yields 2 acetyl-CoA + 2 NADH + 2 H + + 2 CO2

In prokaryotic cells, the transition step occurs in the cytoplasm in eukaryotic cells the pyruvates must first enter the mitochondria because the transition reaction and the citric acid cycle take place in the matrix of the mitochondria.

The two molecules of acetyl-CoA can now enter the citric acid cycle. Acetyl-CoA is also a precursor metabolite for fatty acid synthesis, as shown in Figure (PageIndex<3>).

Figure (PageIndex<3>): Integration of Metabolism - Precursor Metabolites. Carbohydrates, proteins, and lipids can be used as energy sources metabolites involved in energy production can be used to synthesize carbohydrates, proteins, lipids, nucleic acids, and cellular structures.


Mitochondrial and Chloroplastic Targeting Peptides Peptidase, PreP

Elzbieta Glaser , . Pedro Filipe Teixeira , in Handbook of Proteolytic Enzymes (Third Edition) , 2013

Preparation

A mitochondrial matrix fraction isolated from Solanum tuberosum was originally used for isolation and identification of PreP. A three-step purification involving affinity purification with arginine Sepharose and ion-exchange chromatography was used and resulted in a sample that was resolved by 2D-PAGE. Analysis of protein spots by MALDI-TOF mass spectrometry allowed identification of AtPreP1 and AtPreP2 in an A. thaliana database [2] .

To allow a simple and rapid purification procedure, AtPreP1, AtPreP2 and hPreP were expressed in Escherichia coli as fusion to glutathione S-transferase and purified by affinity chromatography [3,6] .


Pyruvate oxidation

Pyruvate oxidation is much shorter than the other steps of cellular respiration, it is key in linking glycolysis and the Kreb’s cycle.

Pyruvate (a 3 carbon molecule) is converted to acetyl CoA, a two-carbon molecule attached to coenzyme A. This reaction releases a molecule of carbon dioxide and reduces a NAD+ to NADH. In eukaryotes, pyruvate oxidation takes place in the matrix, the central compartment of mitochondria. Acetyl-CoA, acts as fuel for the Kreb’s cycle (also called the citric acid cycle). Before the reactions in this process can begin, pyruvate must enter the mitochondrion, passing through its inner membrane and to the matrix. In the matrix, pyruvate is modified in a series of steps:

Firstly, a carboxyl group is removed from pyruvate and released as carbon dioxide. The resulting two carbon molecule is oxidized, and NAD+ acts as the electron acceptor for the lost electrons, forming NADH. The oxidized two-carbon molecule is attached to Coenzyme A to form acetyl CoA. Acetyl CoA carries the acetyl group to the Kreb’s cycle.

These steps are carried out by a large enzyme complex called the pyruvate dehydrogenase complex , which consists of three component enzymes and includes over 60 subunits. The pyruvate dehydrogenase complex is a key target for regulation, as it controls the amount of acetyl-CoA that can enter the Kreb’s cycle. For each glucose molecule, 2 molecules of pyruvate are converted into 2 molecules of acetyl-CoA during pyruvate oxidation, releasing 2 carbons as carbon dioxide and generating 2 NADH from NAD+ . Acetyl-CoA serves as fuel for the Kreb’s cycle in the next stage of cellular respiration.


How is Coenzyme A Transported to the Matrix? - Biology

The Krebs cycle occurs in the matrix of the mitochondrion and is the aerobic phase and requires oxygen.

This is also known as the citric acid cycle or the tricarboxylic acid cycle .

The Krebs cycle is a series of steps catalysed by enzymes in the matrix:

Photo credit: BBC.

• A 2-carbon atoms Acetyl CoA enters the cycle and combines with a 4-carbon compound ( oxaloacetate) to give a 6-carbon compound ( citrate/citric acid ). Coenzyme A is reformed. Cycle turns twice for each original glucose molecule.

• The citrate is then gradually converted back to the 4-carbon oxaloacetate again in a series of small enzyme-controlled steps involving decarboxylation and dehydrogenation . 2 C atoms are released in 2 CO2 molecules and 4 pairs of H atoms are removed.

• The CO2 removed is given off as a waste product. It diffuses rut of the mitochondrion and out of the cell.

• The hydrogens removed are picked up by NAD and another coenzyme called FAD (flavin adenine dinucleotide). 1 FAD and 3 NAD molecules are reduced during each turn of the cycle. H in reduced NAD/FAD will be released in oxidative phosphorylation. The main role of the Krebs cycle in respiration is to generate a pool of reduced hydrogen carriers to pass on to the next stage.

• The regenerated oxaloacetate can combine with another ACoA.

• 1 ATP is produced directly by substrate-level phosphorylation for each ACoA entering the cycle.

• Amino acids and fatty acids can be broken down and fed into cycle.


#87 Respiration, Glycolysis

Respiration is the oxidation of energy-containing organic molecules. The energy released from this process is used to combine ADP with inorganic phosphate to make ATP.


All cells obtain useable energy through respiration. Most cells use carbohydrate, usually glucose , as their fuel. Some cells, such as nerve cells, can only use glucose as their respiratory substrate, but others can use fatty acids , glycerol and amino acids .

Respiration may be aerobic or anaerobic. In both cases, glucose or another respiratory substrate is oxidised.
- In aerobic respiration, oxygen is involved, and the substrate is oxidised completely, releasing much of the energy that it contains.
- In anaerobic respiration, oxygen is not involved, and the substrate is only partially oxidised. Only a small proportion of the energy it contains is released.

Respiration of glucose has 4 main stages:

glycolysis in the cytoplasm (cytosol) of the cell
• the link reaction in the matrix of a mitochondrion
• the Krebs cycle in the matrix of a mitochondrion
oxidative phosphorylation on the inner mitochondrial membrane.

Glycolysis (the breakdown of glucose) is the first stage of respiration. It takes place in the cytoplasm and does not require oxygen. It begins with the 6-carbon ring-shaped structure of a single glucose molecule and ends with 2 molecules of a 3-carbon sugar called pyruvate and a net gain of 2 ATP. Glycolysis is summarised below.

• A glucose (or other hexose sugar) is phosphorylated, using phosphate from 2 molecules of ATP, to give hexose bisphosphate . This phosphorylation converts an energy-rich but unreactive molecule into one that is much more reactive, the chemical potential energy of which can be trapped more efficiently.



• The hexose bisphosphate is split into 2 triose phosphate molecules.

• Hydrogen atoms and phosphate groups are removed from the triose phosphate (by the coenzyme NAD). The removal of hydrogens is an oxidation reaction, so triose phosphate is oxidised to 2 molecules of pyruvate (pyruvic acid). During this step, the phosphate groups from the triose phosphates are added to ADP to produce a small yield of ATP.

• Overall, 2 molecules of ATP are used and 4 are made during glycolysis of one glucose molecule, making a net gain of 2 ATPs per glucose. The pyruvic acid is then converted to either lactic acid or alcohol and carbon dioxide without the production of any more ATP.

If the cell cannot catabolize the pyruvate molecules further, it will harvest only 2 ATP molecules from 1 molecule of glucose. For example, mature mammalian red blood cells are only capable of glycolysis, which is their sole source of ATP. If glycolysis is interrupted, these cells would eventually die.

Coenzymes

Respiration involves coenzymes called NAD and FAD. A coenzyme is a molecule required for an enzyme to be able to catalyse a reaction. Hydrogens removed during glycolysis are transferred to the hydrogen carrier molecule nicotinamide adenine dinucleotide ( NAD ) to give reduced NAD. The term 'reduce' means to add hydrogen, so reduced NAD has had hydrogen added to it. NAD is present in cells in small quantities and is continually recycled.


Mitochondrial Electron Transport Chain

The mitochondrial electron transport chain is composed of three main membrane-associated electron carriers flavoproteins (FMN, FAD), cytochromes, and quinones (coenzyme Q, also known as ubiquinone because it is a ubiquitous quinone in biological systems).

All these electron carriers reside within the inner membrane of the mitochondria and operate together to transfer electrons from donors, like NADH and FADH2, to acceptors, such as O2. The, electrons flow from carriers with more negative reduction potentials to those with more positive reduction potentials and eventually combine with O2 and H to form water.

However, the mitochondrial electron transport system is arranged into four enzyme complexes of carriers, each capable of transporting electrons part of the way to O2 (Fig. 24.5). Coenzyme Q and cytochrome c connect the complexes with each other.

The four enzyme complexes of carriers are: NADH-Q oxidoreductase, succinate-Q-reductase, Q-cytochrome c oxidoreductase, and cytochrome c oxidase. These complexes are the enzyme complex and each of them consists of different prosthetic groups (Table 24.2).

The process of mitochondrial electron transport chain is summarized in Figure 24.6, which shows the flow of electrons and protons through the four enzyme complexes of the transport chain.

The whole process of mitochondrial electron transport can be represented in brief in the following manner:

1. Electrons donated by NADH enter the chain at complex I (NADH-Q-oxidoreductase) and pass through a flavoprotein (FMN) to a series of iron-sulphur-proteins (FeS) and then to ubiquinone (Q).

2. Electrons donated by succinate enter the chain at Complex II (succinate-Q-reductase) and pass through a flavoprotein (FAD) and FeS centres and then to ubiquinone (Q).

3. Ubiquinone (Q) serves as a mobile carrier of electrons received from complexes I and II and passes them to complex III (Q-cytochrome c oxidoreductase).

4. Complex III called Q-cytochrome c oxidoreductase or cytochrome bc1 complex passes the electrons through its prosthetic groups Cyt bL (Heme bL), Cyt bH (heme bH), FeS, and Cyt cL (Heme cL) to cytochrome c.

5. Cytochrome c (Cyt c), a mobile connecting link between complex III and IV, passes electrons to complex IV (cytochrome c oxidase). The latter carries electrons through its prosthetic groups Cyt a (Heme a), Cyt a3 (Heme a3) CuA and CuB and transfers them to molecular oxygen, reducing it to H2O.

6. Electron flow through complexes I, III and IV is accompanied by proton flow from the mitochondrial matrix (which becomes negatively charged) to inter membrane space or cytosolic side (which becomes positively charged). The number of protons (H + ) moved across the membrane at each site per pair of electrons transported is still somewhat uncertain the current consensus is that at least 10 protons move outward during NADH oxidation.


Respiration

Aerobic respiration is made of four stages: glycolysis, the link reaction, the Krebs cycle and oxidative phosphorylation. During aerobic respiration, glucose is effectively burned inside our bodies (it reacts with oxygen) to produce carbon dioxide, water and lots of energy in the form of ATP. The overall equation for aerobic respiration is:

Glycolysis

The first stage of aerobic respiration is glycolysis, which takes place in the cytoplasm. Glycolysis converts glucose, a six-carbon molecule, into two smaller three-carbon molecules called pyruvate. This stage doesn’t require oxygen so it is an anaerobic process and is involved in both aerobic and anaerobic respiration pathways.

Glucose is phosphorylated using the phosphate groups from two molecules of ATP. ATP is hydrolysed into ADP and inorganic phosphate. This forms a molecule which is unstable and immediately breaks down into two three-carbon molecules called triose phosphate (TP). Hydrogen is removed from TP to convert it into pyruvate. The hydrogen is transferred to a coenzyme called NAD to form reduced NAD (NADH). The removal of hydrogen from TP oxidises it. The reduced NAD is used in the last stage of aerobic respiration, oxidative phosphorylation, whereas the pyruvate moves into the mitochondria for the next stage of respiration, the link reaction.

The conversion of triose phosphate to pyruvate produced four molecules of ATP. Since two molecules were used for the phosphorylation of glucose in the first step, this means there is a net gain of two ATP molecules in glycolysis.

The Link Reaction

The link reaction takes place in the mitochondrial matrix.

The link reaction takes place in the mitochondrial matrix and converts pyruvate into a molecule called acetyl coenzyme A (acetyl CoA). This stage does not produce any energy in the form of ATP but does produce reduced NAD and acetyl CoA. Reduced NAD will be used in oxidative phosphorylation while the acetyl CoA will be used in the next stage of aerobic respiration, the Krebs cycle.

During the link reaction, a carbon atom is removed from pyruvate, forming carbon dioxide. This converts pyruvate into a two-carbon molecule called acetate. Hydrogen is also removed from pyruvate in the conversion into acetate, which is picked up by the coenzyme NAD to form reduced NAD. The acetate is combined with coenzyme A (CoA) to form acetyl CoA.

Since one glucose molecule is converted into 2x pyruvate, the link reaction happens twice for every glucose molecule. This means that each molecule of glucose produces two molecules of acetyl CoA (along with 2x carbon dioxide and 2x NADH).

The Krebs cycle

The Kreb’s cycle (also known as the citric acid cycle) is a series of reactions which generate reduced NAD and a similar molecule called reduced FAD which are needed for oxidative phosphorylation. Acetyl CoA from the link reaction reacts with a four-carbon molecule called oxaloacetate. The coenzyme A portion of acetyl CoA is removed and returns to the link reaction to be reused. A 6-carbon molecule called citrate is produced. Carbon and hydrogen are removed from citrate, forming carbon dioxide and reduced NAD. The citrate is converted into a 5-carbon compound. Decarboxylation and dehydrogenation occur once more, which converts the 5-carbon compounds into the 4-carbon molecule oxaloacetate which we started with. ATP, 2 molecules of reduced NAD, one molecule of FAD and carbon dioxide are also formed in this step. This cycle takes place twice for each glucose molecule that is respired aerobically.

Oxidative Phosphorylation

Oxidative phosphorylation is the last stage of aerobic respiration and it is the part where most of the ATP is made. It uses the electrons that are being carried by reduced NAD and reduced FAD that have been generated in the first three stages. It takes place across the inner mitochondrial membrane and involves two processes - the electron transport chain and chemiosmosis.

The coenzymes reduced NAD and reduced FAD release hydrogen atoms which split into hydrogen ions and electrons. The electrons are passed onto electron carriers which are embedded within the inner mitochondrial membrane and travel along a series of electron carriers known as the electron transport chain. As they travel between the electron carriers, they lose energy. This energy is used by the carriers to pump hydrogen ions from the mitochondrial matrix across the inner membrane. Hydrogen ions accumulate in the intermembrane space and this generates a proton gradient (sometimes referred to as an electrochemical gradient) across the membrane. Hydrogen ions then flow back into the matrix through the enzyme ATP synthase which uses the movement of hydrogen ions (the proton motive force) to add a phosphate group onto ADP to form ATP. The process by which the movement of hydrogen ions produces ATP is called chemiosmosis. Once the electrons reach the end of the electron transport chain, they are passed onto oxygen, which is referred to as the ‘final electron acceptor’. Oxygen combines with electrons and hydrogen ions to form water, one of the products of aerobic respiration.

Metabolic poisons, such as cyanide, disrupt oxidative phosphorylation by binding to electron carriers and inhibiting the movement of electrons along the electron transport chain. This reduces chemiosmosis since a proton gradient is not established and also inhibits the Krebs cycle since NAD and FAD are not regenerated. ATP production grounds to a halt so processes which require energy (such as the contraction of heart muscle) cannot take place, which can be deadly for the organism who has ingested the poison.

Total ATP production

Aerobic respiration produces a total of 38 ATP molecules per one molecule of glucose respired. Here’s a breakdown of the ATP production at each of the different stages. Each molecule of reduced NAD produces 3 ATP and each molecule of reduced FAD produces 2 ATP. Remember that the link reaction and Krebs cycle happen twice for each molecule of glucose, because it is converted into 2x pyruvate.

Glycolysis: direct production of 2 ATP

Glycolysis: 2 reduced NAD are converted into 6 ATP (2 x 3) in oxidative phosphorylation

Link reaction: 2 reduced NAD are converted into 6 ATP (2 x 3) in oxidative phosphorylation

Krebs cycle: direct production of 2 ATP

Krebs cycle: 6 reduced NAD are converted into 18 ATP (6 x 3) in oxidative phosphorylation

Krebs cycle: 2 reduced FAD are converted into 4 ATP (2 x 2) in oxidative phosphorylation

Total ATP = 2 + 6 + 6 + 2 + 18 + 4 = 38 ATP

Measuring the rate of respiration

The rate of respiration is measured using a piece of apparatus called a respirometer and works by measuring either the amount of oxygen used up by an organism or the amount of carbon dioxide produced. The faster the amount of oxygen consumed, the faster the rate of respiration.

You would set up the respirometer as shown in the diagram, with respiring organisms (such as woodlice) in one test tube connected to another test tube by a manometer. The manometer contains a coloured liquid which will move closer towards the respiring test tube as oxygen is consumed. The test tube on the right is a control test tube, containing a non-respiring substance, such as glass beads. The purpose of the control tube is to ensure that only respiration is causing the movement of liquid in the manometer. The control tube should be as similar as possible to the test tube e.g. the glass beads should be the same mass as the woodlice. In each test tube you need to add the same volume of potassium hydroxide solution which absorbs carbon dioxide - this ensures that the movement of the liquid is only affected by the decreasing levels of oxygen.

Once the apparatus has been set up, it is left for a certain period of time (e.g. 30 minutes). This will allow for the potassium hydroxide to absorb all of the carbon dioxide in the test tubes. You then record the distance moved by the liquid in the manometer in a given time, using the calibrated scale and a stopwatch. You then calculate the volume of oxygen taken in by the woodlice per minute. Repeat the experiment at least three times and calculate a mean.

Anaerobic respiration

Respiration can also occur in the absence of oxygen - this is called anaerobic respiration. In mammals, glucose can be converted into lactate (aka lactic acid) which releases a small amount of energy in the form of ATP.

The first step of anaerobic respiration is the same as aerobic respiration: glycolysis. Glucose is converted into pyruvate with the net release of 2 ATP molecules. 2 molecules of reduced NAD are also formed. In the second step, reduced NAD donates hydrogen (and electrons) to pyruvate, producing lactate and NAD. This regenerates more oxidised NAD for glycolysis. This enables anaerobic respiration to continue and ensures that small amounts of energy can still be made in the absence of oxygen, allowing biological reactions to keep ticking over.

Continued anaerobic respiration results in the build up of lactate, which needs to be broken down. Cells can convert lactate back into pyruvate, which is then able to enter aerobic respiration at the Krebs cycle. In addition, liver cells have the ability to convert lactate into glucose, which can then be respired aerobically (if oxygen is now present) or stored for later use.


Section Summary

The citric acid cycle is a series of chemical reactions that removes high-energy electrons and uses them in the electron transport chain to generate ATP. One molecule of ATP (or an equivalent) is produced per each turn of the cycle.

The electron transport chain is the portion of aerobic respiration that uses free oxygen as the final electron acceptor for electrons removed from the intermediate compounds in glucose catabolism. The electrons are passed through a series of chemical reactions, with a small amount of free energy used at three points to transport hydrogen ions across the membrane. This contributes to the gradient used in chemiosmosis. As the electrons are passed from NADH or FADH2 down the electron transport chain, they lose energy. The products of the electron transport chain are water and ATP. A number of intermediate compounds can be diverted into the anabolism of other biochemical molecules, such as nucleic acids, non-essential amino acids, sugars, and lipids. These same molecules, except nucleic acids, can serve as energy sources for the glucose pathway.

Exercises

Glossary

acetyl CoA: the combination of an acetyl group derived from pyruvic acid and coenzyme A which is made from pantothenic acid (a B-group vitamin)

ATP synthase: a membrane-embedded protein complex that regenerates ATP from ADP with energy from protons diffusing through it

chemiosmosis: the movement of hydrogen ions down their electrochemical gradient across a membrane through ATP synthase to generate ATP

citric acid cycle: a series of enzyme-catalyzed chemical reactions of central importance in all living cells that harvests the energy in carbon-carbon bonds of sugar molecules to generate ATP the citric acid cycle is an aerobic metabolic pathway because it requires oxygen in later reactions to proceed

electron transport chain: a series of four large, multi-protein complexes embedded in the inner mitochondrial membrane that accepts electrons from donor compounds and harvests energy from a series of chemical reactions to generate a hydrogen ion gradient across the membrane

oxidative phosphorylation: the production of ATP by the transfer of electrons down the electron transport chain to create a proton gradient that is used by ATP synthase to add phosphate groups to ADP molecules