# 4.5: Cellular Respiration

Cellular respiration is the process of oxidizing food molecules, like glucose, to carbon dioxide and water.

$C_6H_{12}O_{6} + 6O_2 + 6H_2O → 12H_2O + 6 CO_2$

The energy released is trapped in the form of ATP for use by all the energy-consuming activities of the cell. The process occurs in two phases:

• glycolysis, the breakdown of glucose to pyruvic acid
• the complete oxidation of pyruvic acid to carbon dioxide and water

In eukaryotes, glycolysis occurs in the cytosol and the remaining processes take place in mitochondria.

### Mitochondria

Mitochondria are membrane-enclosed organelles distributed through the cytosol of most eukaryotic cells. Their number within the cell ranges from a few hundred to, in very active cells, thousands. Their main function is the conversion of the potential energy of food molecules into ATP.

Fig. 4.5.1 Mitochondria                                    Fig. 4.5.2 Mitochondrion from bat pancreas cell courtesy Keith R. Porter

Mitochondria have:

• an outer membrane that encloses the entire structure
• an inner membrane that encloses a fluid-filled matrix
• between the two is the intermembrane space
• the inner membrane is elaborately folded with shelflike cristae projecting into the matrix.
• a small number (some 5–10) circular molecules of DNA

This electron micrograph in Fig. 4.5.2, shows a single mitochondrion from a bat pancreas cell. Note the double membrane and the way the inner membrane is folded into cristae. The dark, membrane-bounded objects above the mitochondrion are lysosomes. The number of mitochondria in a cell can increase either by their fission (e.g. following mitosis) or decrease by their fusing together. Defects in either process can produce serious, even fatal, illness.

#### The Outer Membrane

The outer membrane contains many complexes of integral membrane proteins that form channels through which a variety of molecules and ions move in and out of the mitochondrion.

#### The Inner Membrane

The inner membrane contains 5 complexes of integral membrane proteins:

• NADH dehydrogenase (Complex I)
• succinate dehydrogenase (Complex II)
• cytochrome c reductase (Complex III; also known as the cytochrome b-c1 complex)
• cytochrome c oxidase (Complex IV)
• ATP synthase (Complex V)

### The Matrix

The matrix contains a complex mixture of soluble enzymes that catalyze the respiration of pyruvic acid and other small organic molecules. Here pyruvic acid is

• oxidized by NAD+ producing NADH + H+
• decarboxylated producing a molecule of
• carbon dioxide (CO2) and
• a 2-carbon fragment of acetate bound to coenzyme A forming acetyl-CoA

### The Citric Acid Cycle

Fig. 4.5.3 The Citric Acid Cycle

This 2-carbon fragment is donated to a molecule of oxaloacetic acid. The resulting molecule of citric acid (which gives its name to the process) undergoes the series of enzymatic steps shown in the diagram. The final step regenerates a molecule of oxaloacetic acid and the cycle is ready to turn again. A brief summary of the cycle is as follows:

• Each of the 3 carbon atoms present in the pyruvate that entered the mitochondrion leaves as a molecule of carbon dioxide (CO2).
• At 4 steps, a pair of electrons (2e-) is removed and transferred to NAD+ reducing it to NADH + H+.
• At one step, a pair of electrons is removed from succinic acid and reduces the prosthetic group flavin adenine dinucleotide (FAD) to FADH2.
• The electrons of NADH and FADH2 are transferred to the electron transport chain.

### The Electron Transport Chain

Fig. 4.5.4 Electron Transport Chain

The electron transport chain consists of 3 complexes of integral membrane proteins

• the NADH dehydrogenase complex (I)
• the cytochrome c reductase complex (III)
• the cytochrome c oxidase complex (IV)

and two freely-diffusible molecules ubiquinone, cytochrome c,  that shuttle electrons from one complex to the next.

The electron transport chain accomplishes:

• The stepwise transfer of electrons from NADH (and FADH2) to oxygen molecules to form (with the aid of protons) water molecules (H2O). Cytochrome c can only transfer one electron at a time, so cytochrome c oxidase must wait until it has accumulated 4 of them before it can react with oxygen.
• Harnessing the energy released by this transfer to the pumping of protons (H+) from the matrix to the intermembrane space.
• Approximately 20 protons are pumped into the intermembrane space as the 4 electrons needed to reduce oxygen to water pass through the respiratory chain.
• The gradient of protons formed across the inner membrane by this process of active transport forms a miniature battery.
• The protons can flow back down this gradient only by reentering the matrix through ATP synthase, another complex (complex V) of 16 integral membrane proteins in the inner membrane. The process is called chemiosmosis.

### Chemiosmosis in mitochondria

Fig.4.5.5 Chemiosmosis in Mitochondria

The energy released as electrons pass down the gradient from NADH to oxygen is harnessed by three enzyme complexes of the respiratory chain (I, III, and IV) to pump protons (H+) against their concentration gradient from the matrix of the mitochondrion into the intermembrane space.

As their concentration increases there (which is the same as saying that the pH decreases), a strong diffusion gradient is set up. The only exit for these protons is through the ATP synthase complex. As in chloroplasts, the energy released as these protons flow down their gradient is harnessed to the synthesis of ATP. The process is called chemiosmosis and is an example of facilitated diffusion. One-half of the 1997 Nobel Prize in Chemistry was awarded to Paul D. Boyer and John E. Walker for their discovery of how ATP synthase works.

### How many ATPs?

It is tempting to try to view the synthesis of ATP as a simple matter of stoichiometry (the fixed ratios of reactants to products in a chemical reaction). But (with 3 exceptions) it is not. Most of the ATP is generated by the proton gradient that develops across the inner mitochondrial membrane. The number of protons pumped out as electrons drop from NADH through the respiratory chain to oxygen is theoretically large enough to generate, as they return through ATP synthase, 3 ATPs per electron pair (but only 2 ATPs for each pair donated by FADH2).

With 12 pairs of electrons removed from each glucose molecule,

• 10 by NAD+ (so 10x3=30); and
• 2 by FADH2 (so 2x2=4),

this could generate 34 ATPs. Add to this the 4 ATPs that are generated by the 3 exceptions and one arrives at 38. However, the energy stored in the proton gradient is also used for the active transport of several molecules and ions through the inner mitochondrial membrane into the matrix. NADH is also used as reducing agent for many cellular reactions. So the actual yield of ATP as mitochondria respire varies with conditions and probably seldom exceeds 30.

#### The three exceptions

A stoichiometric production of ATP does occur at:

• one step in the citric acid cycle yielding 2 ATPs for each glucose molecule. This step is the conversion of alpha-ketoglutaric acid to succinic acid.
• at two steps in glycolysis yielding 2 ATPs for each glucose molecule.

### Mitochondrial DNA (mtDNA)

The human mitochondrion contains 5–10 identical, circular molecules of DNA. Each consists of 16,569 base pairs carrying the information for 37 genes which encode:

• 2 different molecules of ribosomal RNA (rRNA)
• 22 different molecules of transfer RNA (tRNA) (at least one for each amino acid)
• 13 polypeptides

The rRNA and tRNA molecules are used in the machinery that synthesizes the 13 polypeptides.

The 13 polypeptides participate in building several protein complexes embedded in the inner mitochondrial membrane.

• 7 subunits that make up the mitochondrial NADH dehydrogenase (complex I)
• cytochrome b, a subunit of cytochrome c reductase (complex III)
• 3 subunits of cytochrome c oxidase (complex IV)
• 2 subunits of ATP synthase (complex V)

Each of these protein complexes also requires subunits that are encoded by nuclear genes, synthesized in the cytosol, and imported from the cytosol into the mitochondrion. Nuclear genes also encode ~1,000 other proteins that must be imported into the mitochondrion.

#### Mutations in mtDNA cause human diseases

Mutations in 12 of the 13 polypeptide-encoding mitochondrial genes have been found to cause human disease. Although many different organs may be affected, disorders of the muscles and brain are the most common. Perhaps this reflects the great demand for energy of both these organs. (Although representing only ~2% of our body weight, the brain consumes ~20% of the energy produced when we are at rest.)

Some of these disorders are inherited in the germline. In every case, the mutant gene is received from the mother because none of the mitochondria in sperm survives in the fertilized egg. Other disorders are somatic; that is, the mutation occurs in the somatic tissues of the individual.

Example: Exercise Intolerance

A number of humans who suffer from easily-fatigued muscles turn out to have a mutations in their cytochrome b gene. Curiously, only the mitochondria in their muscles have the mutation; the mtDNA of their other tissues is normal. Presumably, very early in their embryonic development, a mutation occurred in a cytochrome b gene in the mitochondrion of a cell destined to produce their muscles.

The severity of mitochondrial diseases varies greatly. The reason for this is probably the extensive mixing of mutant DNA and normal DNA in the mitochondria as they fuse with one another. A mixture of both is called heteroplasmy. The higher the ratio of mutant to normal, the greater the severity of the disease. In fact by chance alone, cells can on occasion end up with all their mitochondria carrying all-mutant genomes — a condition called homoplasmy (a phenomenon resembling genetic drift).

#### Mitochondrial Replacement Techniques

As I noted above, only mothers can pass mutant mtDNA on to their offspring. Two techniques are under intense investigation, either of which could enable a mother to have children free of defective mitochondria. Mutations in some 228 nuclear genes have also been implicated in human mitochondrial diseases, but mitochondrial replacement techniques will not be able to help with these.

#### Why do mitochondria have their own genome?

Many of the features of the mitochondrial genetic system resemble those found in bacteria. This has strengthened the theory that mitochondria are the evolutionary descendants of a bacterium that established an endosymbiotic relationship with the ancestors of eukaryotic cells early in the history of life on earth. However, many of the genes needed for mitochondrial function have since moved to the nuclear genome. The recent sequencing of the complete genome of Rickettsia prowazekii has revealed a number of genes closely related to those found in mitochondria. Perhaps rickettsias are the closest living descendants of the endosymbionts that became the mitochondria of eukaryotes.