Review of cellular respiration

Let’s think back to the metabolism unit of BIOL 1403, where we discussed cellular respiration and photosynthesis.

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

The equation of cellular respiration is: C₆H₁₂O₆ + 6O₂ → 6CO₂ + 6H₂O.

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.

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 23.2, 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 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. Note that the citric acid cycle is also referred to as Krebs cycle or the TCA (tricarboxylic acid) cycle.

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.

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

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.

Fun fact!

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.

Now that we have reviewed the complex process of cellular respiration responsible for producing

ATP, let’s come back to the physiological aspects of cellular respiration, where the focus will be on the respiratory system.

Introducing the respiratory system

Examine the cellular respiration equation again: C₆H₁₂O₆ + 6O₂ → 6CO₂ + 6H₂O.

Recall that the production of ATP using the process of chemiosmosis in mitochondria is called oxidative phosphorylation. The overall result of these reactions is the production of ATP from the energy of the electrons removed from hydrogen atoms. These atoms were originally part of a glucose molecule. At the end of the pathway, the electrons are used to reduce an oxygen molecule to oxygen ions. The extra electrons on the oxygen attract hydrogen ions (protons) from the surrounding medium, and water is formed. Thus, oxygen is the final electron acceptor in the electron transport chain.

But how do humans obtain oxygen and what structures are involved in gas exchange? The key player is the lungs and the respiratory system, which are specifically designed for efficient gas exchange via tiny structures called the alveoli. In this section, we will explore the respiratory system in great detail to obtain a greater appreciation for this incredible organ system. In short, the lungs inspire (inspiration is also referred to as inhalation) oxygen and expire (expiration is also referred to as exhalation) carbon dioxide as a byproduct of cellular respiration. This exchange of oxygen and carbon dioxide is referred to as respiration.

Breathing is an involuntary event. How often a breath is taken and how much air is inhaled or exhaled are tightly regulated by the respiratory center in the brain. Humans, when they aren’t exerting themselves, breathe approximately 15 times per minute on average. Canines, like the dog in Figure 39.1, have a respiratory rate of about 15–30 breaths per minute. With every inhalation, air fills the lungs, and with every exhalation, air rushes back out. That air is doing more than just inflating and deflating the lungs in the chest cavity. The air contains oxygen that crosses the lung tissue, enters the bloodstream, and travels to organs and tissues. Oxygen (O₂) enters the cells where it is used for metabolic reactions that produce ATP, a high-energy compound. At the same time, these reactions release carbon dioxide (CO₂) as a by-product. CO₂ is toxic and must be eliminated. Carbon dioxide exits the cells, enters the bloodstream, travels back to the lungs, and is expired out of the body during exhalation.

Exploring lung anatomy and function

The primary function of the respiratory system is to deliver oxygen to the cells of the body’s tissues and remove carbon dioxide, a cell waste product. The main structures of the human respiratory system are the nasal cavity, the trachea, and lungs.

All aerobic organisms require oxygen to carry out their metabolic functions. Along the evolutionary tree, different organisms have devised different means of obtaining oxygen from the surrounding atmosphere. The environment in which the animal lives greatly determines how an animal respires. The complexity of the respiratory system is correlated with the size of the organism. As animal size increases, diffusion distances increase and the ratio of surface area to volume drops. In unicellular organisms, diffusion across the cell membrane is sufficient for supplying oxygen to the cell (Figure 23.6). Diffusion is a slow, passive transport process. In order for diffusion to be a feasible means of providing oxygen to the cell, the rate of oxygen uptake must match the rate of diffusion across the membrane. In other words, if the cell were very large or thick, diffusion would not be able to provide oxygen quickly enough to the inside of the cell. Therefore, dependence on diffusion as a means of obtaining oxygen and removing carbon dioxide remains feasible only for small organisms or those with highly-flattened bodies, such as many flatworms (Platyhelminthes). Larger organisms had to evolve specialized respiratory tissues, such as gills, lungs, and respiratory passages accompanied by complex circulatory systems, to transport oxygen throughout their entire body.

Direct Diffusion

For small multicellular organisms, diffusion across the outer membrane is sufficient to meet their oxygen needs. Gas exchange by direct diffusion across surface membranes is efficient for organisms less than 1 mm in diameter. In simple organisms, such as cnidarians and flatworms, every cell in the body is close to the external environment. Their cells are kept moist and gases diffuse quickly via direct diffusion. Flatworms are small, literally flat worms, which ‘breathe’ through diffusion across the outer membrane (Figure 23.7). The flat shape of these organisms increases the surface area for diffusion, ensuring that each cell within the body is close to the outer membrane surface and has access to oxygen. If the flatworm had a cylindrical body, then the cells in the center would not be able to get oxygen.

Skin and Gills

Earthworms and amphibians use their skin (integument) as a respiratory organ. A dense network of capillaries lies just below the skin and facilitates gas exchange between the external environment and the circulatory system. The respiratory surface must be kept moist in order for the gases to dissolve and diffuse across cell membranes.

Organisms that live in water need to obtain oxygen from the water. Oxygen dissolves in water but at a lower concentration than in the atmosphere. The atmosphere has roughly 21 percent oxygen. In water, the oxygen concentration is much lower than that. Fish and many other aquatic organisms have evolved gills to take up the dissolved oxygen from water (Figure 23.8). Gills are thin tissue filaments that are highly branched and folded. When water passes over the gills, the dissolved oxygen in water rapidly diffuses across the gills into the bloodstream. The circulatory system can then carry the oxygenated blood to the other parts of the body. In animals that contain coelomic fluid instead of blood, oxygen diffuses across the gill surfaces into the coelomic fluid. Gills are found in mollusks, annelids, and crustaceans.

The folded surfaces of the gills provide a large surface area to ensure that the fish gets sufficient oxygen. Diffusion is a process in which material travels from regions of high concentration to low concentration until equilibrium is reached. In this case, blood with a low concentration of oxygen molecules circulates through the gills. The concentration of oxygen molecules in water is higher than the concentration of oxygen molecules in gills. As a result, oxygen molecules diffuse from water (high concentration) to blood (low concentration), as shown in Figure 23.9. Similarly, carbon dioxide molecules in the blood diffuse from the blood (high concentration) to water (low concentration).

Tracheal Systems

Insect respiration is independent of its circulatory system; therefore, the blood does not play a direct role in oxygen transport. Insects have a highly specialized type of respiratory system called the tracheal system, which consists of a network of small tubes that carries oxygen to the entire body. The tracheal system is the most direct and efficient respiratory system in active animals. The tubes in the tracheal system are made of a polymeric material called chitin.

Insect bodies have openings, called spiracles, along the thorax and abdomen. These openings connect to the tubular network, allowing oxygen to pass into the body (Figure 23.10) and regulating the diffusion of CO₂ and water vapor. Air enters and leaves the tracheal system through the spiracles. Some insects can ventilate the tracheal system with body movements.

Mammalian Systems

In mammals, pulmonary ventilation occurs via inhalation (breathing). During inhalation, air enters the body through the nasal cavity located just inside the nose (Figure 29.11). As air passes through the nasal cavity, the air is warmed to body temperature and humidified. The respiratory tract is coated with mucus to seal the tissues from direct contact with air. Mucus is high in water. As air crosses these surfaces of the mucous membranes, it picks up water. These processes help equilibrate the air to the body conditions, reducing any damage that cold, dry air can cause. Particulate matter that is floating in the air is removed in the nasal passages via mucus and cilia. The processes of warming, humidifying, and removing particles are important protective mechanisms that prevent damage to the trachea and lungs. Thus, inhalation serves several purposes in addition to bringing oxygen into the respiratory system.

From the nasal cavity, air passes through the pharynx (throat) and the larynx (voice box), as it makes its way to the trachea (Figure 23.11). The main function of the trachea is to funnel the inhaled air to the lungs and the exhaled air back out of the body. The human trachea is a cylinder about 10 to 12 cm long and 2 cm in diameter that sits in front of the esophagus and extends from the larynx into the chest cavity where it divides into the two primary bronchi at the midthorax. It is made of incomplete rings of hyaline cartilage and smooth muscle (Figure 23.12). The trachea is lined with mucus-producing goblet cells and ciliated epithelia. The cilia propel foreign particles trapped in the mucus toward the pharynx. The cartilage provides strength and support to the trachea to keep the passage open. The smooth muscle can contract, decreasing the trachea’s diameter, which causes expired air to rush upwards from the lungs at a great force. The forced exhalation helps expel mucus when we cough. Smooth muscle can contract or relax, depending on stimuli from the external environment or the body’s nervous system.

Lungs: Bronchi and Alveoli

The end of the trachea bifurcates (divides) to the right and left lungs. The lungs are not identical. The right lung is larger and contains three lobes, whereas the smaller left lung contains two lobes (Figure 23.13). The muscular diaphragm, which facilitates breathing, is inferior to (below) the lungs and marks the end of the thoracic cavity.

In the lungs, air is diverted into smaller and smaller passages, or bronchi. Air enters the lungs through the two primary (main) bronchi (singular: bronchus). Each bronchus divides into secondary bronchi, then into tertiary bronchi, which in turn divide, creating smaller and smaller diameter bronchioles as they split and spread through the lung. Like the trachea, the bronchi are made of cartilage and smooth muscle. At the bronchioles, the cartilage is replaced with elastic fibers. Bronchi are innervated by nerves of both the parasympathetic and sympathetic nervous systems that control muscle contraction (parasympathetic) or relaxation (sympathetic) in the bronchi and bronchioles, depending on the nervous system’s cues. In humans, bronchioles with a diameter smaller than 0.5 mm are the respiratory bronchioles. They lack cartilage and therefore rely on inhaled air to support their shape. As the passageways decrease in diameter, the relative amount of smooth muscle increases.

The terminal bronchioles subdivide into microscopic branches called respiratory bronchioles. The respiratorybronchioles subdivide into several alveolar ducts. Numerous alveoli and alveolar sacs surround the alveolar ducts. The alveolar sacs resemble bunches of grapes tethered to the end of the bronchioles (Figure 23.14). In the acinar region, the alveolar ducts are attached to the end of each bronchiole. At the end of each duct are approximately 100 alveolar sacs, each containing 20 to 30 alveoli that are 200 to 300 microns in diameter. Gas exchange occurs only in alveoli. Alveoli are made of thin-walled parenchymal cells, typically one-cell thick, that look like tiny bubbles within the sacs. Alveoli are in direct contact with capillaries (one-cell thick) of the circulatory system. Such intimate contact ensures that oxygen will diffuse from alveoli into the blood and be distributed to the cells of the body. In addition, the carbon dioxide that was produced by cells as a waste product will diffuse from the blood into alveoli to be exhaled. The anatomical arrangement of capillaries and alveoli emphasizes the structural and functional relationship of the respiratory and circulatory systems. Because there are so many alveoli (~300 million per lung) within each alveolar sac and so many sacs at the end of each alveolar duct, the lungs have a sponge-like consistency. This organization produces a very large surface area that is available for gas exchange. The surface area of alveoli in the lungs is approximately 75 m². This large surface area, combined with the thin-walled nature of the alveolar parenchymal cells, allows gases to easily diffuse across the cells.

Protective Mechanisms

The air that organisms breathe contains particulate matter such as dust, dirt, viral particles, and bacteria that can damage the lungs or trigger allergic immune responses. The respiratory system contains several protective mechanisms to avoid problems or tissue damage. In the nasal cavity, hairs and mucus trap small particles, viruses, bacteria, dust, and dirt to prevent their entry.

If particulates do make it beyond the nose, or enter through the mouth, the bronchi and bronchioles of the lungs also contain several protective devices. The lungs produce mucus—a sticky substance made of mucin, a complex glycoprotein, as well as salts and water—that traps particulates. The bronchi and bronchioles contain cilia, small hair-like projections that line the walls of the bronchi and bronchioles (Figure 23.15). These cilia beat in unison and move mucus and particles out of the bronchi and bronchioles back up to the throat where it is swallowed and eliminated via the esophagus.

In humans, for example, tar and other substances in cigarette smoke destroy or paralyze the cilia, making the removal of particles more difficult. In addition, smoking causes the lungs to produce more mucus, which the damaged cilia are not able to move. This causes a persistent cough, as the lungs try to rid themselves of particulate matter, and makes smokers more susceptible to respiratory ailments.

Metabolomics

Metabolomics is the scientific study of chemical processes involving metabolites. The metabolome represents the collection of all metabolites, which are the end products of cellular processes, in a biological cell, tissue, organ, or organism. Thus, while mRNA gene expression data and proteomic analyses do not tell the whole story of what might be happening in a cell, metabolic profiling can give an instantaneous snapshot of the physiology of that cell. One of the challenges of systems biology and functional genomics is to integrate proteomic, transcriptomic, and metabolomic information to give a more complete picture of living organisms.

History and Development

The idea that biological fluids reflect the health of an individual has existed for a long time. The term “metabolic profile” was introduced by Horning, et al. in 1971, after they demonstrated that gas chromatography- mass spectrometry (GC-MS; ) could be used to measure compounds present in human urine and tissue extracts. GC-MS is a method that combines the features of gas-liquid chromatography and mass spectrometry to identify different substances within a test sample. Concurrently, NMR spectroscopy, which was discovered in the 1940s, was also undergoing rapid advances. In 1974, Seeley et al. demonstrated the utility of using NMR to detect metabolites in unmodified biological samples. This first study on muscle tissue highlighted the value of NMR, in that it was determined that 90% of cellular ATP is complexed with magnesium. As sensitivity has improved with the evolution of higher magnetic field strengths and magic-angle spinning, NMR continues to be a leading analytical tool to investigate metabolism.

 

In 2005, the first metabolomics web database for characterizing human metabolites, METLIN, was developed in the Siuzdak laboratory at The Scripps Research Institute. METLIN contained over 10,000 metabolites and tandem mass spectral data. On January 23, 2007, the Human Metabolome Project, led by Dr. David Wishart of the University of Alberta, Canada, completed the first draft of the human metabolome, consisting of a database of approximately 2500 metabolites, 1200 drugs and 3500 food components.

As late as mid-2010, metabolomics was still considered an “emerging field”. Further, it was noted that further progress in the field was in large part the result of addressing otherwise “irresolvable technical challenges” through technical evolution of mass spectrometry instrumentation. The word was coined in analogy with transcriptomics and proteomics. Like the transcriptome and the proteome, the metabolome is dynamic, changing from second to second. Although the metabolome can be defined readily enough, it is not currently possible to analyse the entire range of metabolites by a single analytical method.

Metabolites are the intermediates and products of metabolism. Within the context of metabolomics, a metabolite is usually defined as any molecule less than 1 kDa in size. However, there are exceptions to this, depending on the sample and detection method. Macromolecules such as lipoproteins and albumin are reliably detected in NMR-based metabolomics studies of blood plasma. In plant-based metabolomics, it is common to refer to “primary metabolites,” which are directly involved in growth, development and reproduction, and “secondary metabolites,” which are indirectly involved in growth, development and reproduction. In contrast, in human-based metabolomics it is more common to describe metabolites as being either endogenous (produced by the host organism) or exogenous. The metabolome forms a large network of metabolic reactions, where outputs from one enzymatic chemical reaction are inputs to other chemical reactions. Such systems have been described as hypercycles.

Separation methods: Gas chromatography, especially when interfaced with mass spectrometry (GC-MS), is one of the most widely used and powerful methods. It offers very high chromatographic resolution, but requires chemical derivatization for many biomolecules: only volatile chemicals can be analysed without derivatization.

Detection methods: Mass spectrometry (MS) is used to identify and to quantify metabolites after separation. Surface-based mass analysis has seen a resurgence in the past decade, with new MS technologies focused on increasing sensitivity, minimizing background, and reducing sample preparation.

Statistical methods: The data generated in metabolomics usually consist of measurements performed on subjects under various conditions. These measurements may be digitized spectra, or a list of metabolite levels. In its simplest form this generates a matrix with rows corresponding to subjects and columns corresponding to metabolite levels.

Key applications

• Toxicity assessment/toxicology. Metabolic profiling, especially of urine or blood plasma samples, can be used to detect the physiological changes caused by toxic insult of a chemical or mixture of chemicals. This is of particular relevance to pharmaceutical companies wanting to test the toxicity of potential drug candidates.
• Functional genomics. Metabolomics can be an excellent tool for determining the phenotype caused by a genetic manipulation, such as gene deletion or insertion. Sometimes this can be a sufficient goal in itself—for instance, to detect any phenotypic changes in a genetically-modified plant intended for human or animal consumption. More exciting is the prospect of predicting the function of unknown genes by comparison with the metabolic perturbations caused by deletion/insertion of known genes.

References

Adapted from Clark, M.A., Douglas, M., and Choi, J. (2018). Biology 2e. OpenStax. Retrieved from https://openstax.org/books/biology-2e/pages/1-introduction

Kimball, J. W. (n.d.). Biology. LibreTexts Biology. Retrieved from https://bio.libretexts.org/Bookshelves/Introductory_and_General_Biology/Book%3A_Biology_(Kimball)/04%3A_Cell_Metabolism/4.05%3A_Cellular_Respiration

Boundless. (n.d.). Microbiology. LibreTexts Biology. Retrieved from https://bio.libretexts.org/Bookshelves/Microbiology/Microbiology_(Boundless)/07%3A_Microbial_Genetics/7.22%3A_Genomics_and_Proteomics/7.22C%3A__Metabolomics