Reactive Oxygen Species (ROS)

Reactive oxygen species are

molecules like hydrogen peroxide (#5)
ions like the hypochlorite ion (#6)
radicals like the hydroxyl radical (#3). It is the most reactive of them all; note how it differs from the hydroxyl ion (#4).
the superoxide anion (#2) which is both ion and radical.

A radical (also called a "free radical") is a clusters of atoms one of which contains an unpaired electron (shown in red) in its outermost shell of electrons. This is an extremely unstable configuration, and radicals quickly react with other molecules or radicals to achieve the stable configuration of 4 pairs of electrons in their outermost shell (one pair for hydrogen).

Link to discussion of electron organization in atoms.

ROS Formation

Reactive oxygen species are formed by several different mechanisms:

the interaction of ionizing radiation with biological molecules
as an unavoidable byproduct of cellular respiration. Some electrons passing "down" the electron transport chain leak away from the main path (especially as they pass through ubiquinone) and go directly to reduce oxygen molecules to the superoxide anion (#2 above).
synthesized by dedicated enzymes in phagocytic cells like neutrophils and macrophages
- NADPH oxidase (in both type of phagocytes)
- myeloperoxidase (in neutrophils only)

ROS Activity

Strong oxidants like the various ROS can damage other molecules and the cell structures of which they are a part.

Among the most important of these are the actions of free radicals on the fatty acid side chains of lipids in the various membranes of the cell, especially mitochondrial membranes (which are directly exposed to the superoxide anions produced during cellular respiration).
The figure shows one common series of reactions.

A hydroxyl radical removes a hydrogen atom from one of the carbon atoms in the fatty acid chain (only a portion of which is shown) forming
a molecule of water and leaving the carbon atom with an unpaired electron (in red); thus now a radical.
Several possible fates await it. One of the most likely (and shown here) is to react with a molecule of oxygen (O₂) forming a peroxyl radical.
This might then steal a hydrogen atom from a nearby side chain making it now a radical.
One of the insidious things about free radicals is that in interacting with other molecules to gain a stable configuration of electrons, they convert that target molecule into a radical. So a chain reaction begins that will propagate until two radicals meet each other and each contributes its unpaired electron to form a covalent bond linking the two.
Two common examples: The peroxyl radical may interact with:
- another peroxyl radical on a nearby side chain crosslinking them with a covalent bond.
- another nearby carbon-centered radical crosslinking them covalently.
In both these latter cases, radical formation comes to an end but with the result that the fatty acid side chains of membrane lipids may have become so deformed as to damage the membrane.
The lipofuscin so characteristic of aging cells may be formed by these mechanisms [Link].

Defenses Against ROS

Cells have a variety of defenses against the harmful effects of ROS.

These include two enzymes:

superoxide dismutase (SOD), which converts two superoxide anions into a molecule of hydrogen peroxide and one of oxygen, and
catalase

as well as several small molecules that are antioxidants, such as

alpha-tocopherol (vitamin E). This can break the covalent links that ROS have formed between fatty acid side chains in membrane lipids.
uric acid. (Perhaps the long life span of some reptiles and birds is attributable to their high levels of uric acid.)
vitamin C (in the right concentration)

Pharmacy shelves are filled with antioxidant preparations that people take in the hope of warding off the damaging effects (perhaps including aging) of ROS.

ROS are Essential

But it is important that the attempt to limit the production of ROS not succeed too well. Because ROS have important functions to perform in the cell.
Examples:

The cells of the thyroid gland must make hydrogen peroxide in order to attach iodine atoms to thyroglobulin in the synthesis of thyroxine.
Macrophages and neutrophils must generate ROS in order to kill some types of bacteria that they engulf by phagocytosis.
- Bacteria are engulfed into a phagosome.
- This fuses with a lysosome.
- Subunits of the enzyme NADPH oxidase assemble in the lysosome membrane forming the active enzyme.
- It catalyzes the synthesis of the superoxide anion.
  NADPH − 2 e^- + 2O₂ -> NADP⁺ + H⁺ + 2 ^.O₂⁻
- This activity produces a large increase in oxygen consumption, called the "respiratory burst".
- Superoxide dismutase (SOD) converts this into hydrogen peroxide, which kills off the engulfed bacteria (except those that manufacture enough catalase to protect themselves).
Neutrophils (but not macrophages) also kill off engulfed pathogens by using the enzyme myeloperoxidase which catalyzes the reaction of hydrogen peroxide (made from superoxide anions) with chloride ions to produce the strongly antiseptic hypochlorite ion (OCl⁻, #6 above).
H₂O₂ + Cl⁻ -> HOCl (hypochlorous acid) + OH⁻
HOCl -> H⁺ + OCl⁻

Chronic Granulomatous Disease (CGD)

This rare genetic disorder demonstrates the importance of ROS in protecting us from many type of bacterial infection. It is caused by a defective gene for one of the subunits of NADPH oxidase.
People with CGD have a difficult time ridding themselves of bacterial infections — especially those caused by bacteria (e.g. staphylococci, Salmonella) and fungi (e.g., Aspergillus) that produce catalase to protect themselves against the hydrogen peroxide generated by the macrophages and neutrophils that engulf them. Often the result is the development of a persisting nest of infected cells — called a granuloma. The gene for one of the subunits of NADPH most frequently mutated in CGD is on the X chromosome. Thus males are principally affected.
However, examination of the neutrophils of females who are carriers of the gene shows that 50% of them do not make active NADPH oxidase when they engulf pathogens. In these cells, the X chromosome with the nonmutant allele has been inactivated and converted into a Barr body. [Link to discussion]
In June 2005, two cases of successful gene therapy for CGD were reported. Blood stem cells from the patients were removed, and the active gene for the NADPH subunit inserted into them using a retroviral vector. The transformed cells were returned to the patients, took up residence in their bone marrow, proliferated successfully, and improved their symptoms.

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Friday, February 25, 2011