4.05: Cellular Respiration

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.

A simplified schematic diagram of a single mitochondrion, drawn as a horizontal oval with a double membrane. The outermost boundary is the outer membrane (labeled at upper left, with a pointer line to the outer edge). Just inside it runs the inner membrane (labeled at upper right). The narrow gap between the two membranes is the intermembrane space (labeled at lower left). The interior enclosed by the inner membrane is shaded orange and labeled 'Matrix' (label placed in the center). Two shelf-like folds of the inner membrane project inward???one from the top edge and one from the bottom edge???into the orange matrix; one fold is labeled 'Crista' (label at right). The diagram identifies the four structural regions of a mitochondrion: outer membrane, intermembrane space, inner membrane, and matrix, and shows the cristae as infoldings of the inner membrane projecting into the matrix. An elongated organelle lies diagonally across the frame. A smooth outer boundary surrounds many narrow folds that extend across its interior. Small dense granules occur among the folds. Additional membrane layers and dark particles surround the mitochondrion. No labels or scale bar are present.

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 Figure $4.5.1$, 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 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

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.

Flow diagram of the citric acid cycle, titled 'The Citric Acid Cycle' at center. At top, Pyruvic acid (noted as 'from glycolysis out in the cytosol') is converted to Acetyl-CoA, releasing CO2 (shown in blue) while NAD+ is reduced to NADH + H+ (shown in red). Acetyl-CoA then feeds into the cycle at Oxaloacetic acid. The cycle runs clockwise: Oxaloacetic acid ??? Citric acid ??? Isocitric acid ??? alpha-ketoglutaric acid ??? Succinic acid ??? Fumaric acid ??? Malic acid ??? back to Oxaloacetic acid. CO2 (blue) is released at two steps within the cycle: Isocitric acid to alpha-ketoglutaric acid, and alpha-ketoglutaric acid to Succinic acid. NAD+ is reduced to NADH + H+ (red) at three steps: Isocitric to alpha-ketoglutaric, alpha-ketoglutaric to Succinic, and Malic to Oxaloacetic acid. In the Succinic acid to Fumaric acid step, FAD is reduced to FADH2 (labeled 'FADH2 ??? FAD').

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

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.

A schematic of the electron transport chain across the inner mitochondrial membrane. The intermembrane space is on the left and the matrix on the right. Electrons from the Citric Acid Cycle enter as NADH via NADH dehydrogenase and as FADH2; both deliver 2e??? to ubiquinone. Ubiquinone transfers 2e??? to cytochrome c reductase, which passes electrons one at a time (e???) to cytochrome c. Cytochrome c transfers them to cytochrome c oxidase. At these complexes, H+ is pumped from the matrix into the intermembrane space, shown by left-pointing red arrows. Cytochrome c oxidase combines 4e??? and 4H+ with O2 to produce 2H2O. Readable labels include NAD+, NADH dehydrogenase, FADH2, ubiquinone, cytochrome c reductase, cytochrome c, cytochrome c oxidase, O2, H2O, 2e???, e???, H+, and 4H+.

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

Diagram of chemiosmosis in mitochondria. A horizontal pink band labeled 'Inner mitochondrial membrane' separates the 'Intermembrane space' above from the 'Matrix' below. Four protein complexes span the membrane, from left to right: NADH dehydrogenase (green), cytochrome c reductase (red), cytochrome c oxidase (blue), and ATP synthase (yellow). The first three complexes each display an upward arrow labeled 'H+', indicating that protons are pumped from the matrix into the intermembrane space. At ATP synthase on the right, a downward arrow labeled 'H+' shows protons flowing back from the intermembrane space into the matrix through that complex. Beneath ATP synthase in the matrix, a curved arrow leads from 'ADP' to 'ATP', showing that the downhill proton flow through ATP synthase powers the synthesis of ATP from ADP.

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.

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.