ISIS Report 17/06/09

Water and Fire

The Body Does Burn Water

Burning water is a way of life for all organisms, in animals as in plants, with a little help from sunlight perhaps Dr. Mae-Wan Ho

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Can organisms like animals and humans burn water? Burning or oxidising water is what green plants do with the greatest of ease. They depend on absorbing energetic photons from sunlight to make water give up electrons. A great deal of current effort ia dedicated to artificial photosynthesis [1] (Harvesting Energy from Sunlight with Artificial Photosynthesis, SiS 43), and oxidising water (H₂O) to oxygen (O₂) is certainly half of the reaction; the other half is the production of hydrogen (H₂) that can actually be burnt as fuel.

But can other organisms such as animals and human beings do the same? Not in that way, but they burn water all the same. They oxidize water, not to get energy as much as to create exquisitely targeted local weapons for killing bacteria and other microbes. (The recent discovery that the water in cells and tissues of living organisms may be charged up by exposure to infrared light suggest that animals too, may depend on sunlight to energize themselves [2] (Water Electric, SiS 43). I have often wondered why I feel so energetic after sunbathing.)

Antibodies that burn water

Some years ago, a team of Californian researchers discovered the surprising ability of antibodies produced by the immune system to create highly reactive chemicals that cells can use to cleanse themselves and poison invaders, by burning or oxidizing water. Chemists Richard Lerner and Paul Wentworth at the Scripps Research institute of La Jolla, stumbled upon this new role of antibodies by accident [3]. They found antibodies generating H₂O₂ (hydrogen peroxide) when exposed to UV light. They tested numerous other antibodies and the result was the same each time. The antibodies were generating H₂O₂ from singlet oxygen (¹O₂), an energetic and highly reactive form of the oxygen molecule produced when a source of energy, such as UV light excites the ordinary triplet oxygen (³O₂), catalyzed by the antibody.

Singlet oxygen and hydrogen peroxide belong to a class of molecules called reactive oxygen species (ROS) that have become the subject of much research effort in recent years (see Box).

The difference in energy between triplet and singlet oxygen is about 1 eV, well below the energy of the UV photon absorbed by the protein, which can then be transferred to the triplet oxygen. But where do the electrons come from to reduce singlet oxygen to H₂O₂? At first, the researchers thought the reaction was burning the protein itself to produce H₂O₂, but far too much H₂O₂ was produced for that to be the case. The production of H₂O₂ by antibodies was sustained for long periods under UV light, remaining linear up to more than 40 mol equivalent of H₂O₂. When the H₂O₂ was removed, the antibodies resumed H₂O₂ production at the same initial rates, and remained practically unchanged even after 10 cycles of UV irradiation followed by removal of H₂O₂ [4].

After eliminating other sources for the hydrogen peroxide, such as Cl^- and metal ions, the team hit on the idea that the singlet oxygen was reacting with, or burning water to give H₂O₂. In the presence of singlet oxygen, UV irradiation of antibodies leads to incorporation of oxygen from water into H₂O₂. By tagging water with a heavy isotope of oxygen, Lerner’s team confirmed that oxygen in H₂O₂ did indeed come from water, in the ratio of about 1:2.2.relative to molecular oxygen according to the scheme:

2 ¹O₂   +  2 H₂O ->  2H₂O₂  +  ³O₂                                   (1)

The Scripps researchers teamed up with reaction modelling expert William Goddard III of the California Institute of Technology in Pasadena and his student Xin Xu. They suggested that a water molecule could combine with the singlet oxygen to produce hydrogen trioxide (H₂O₃), which eventually rearranges to produce H₂O₂.

Because of energy barriers, the initial reaction of water and singlet oxygen probably would never take place on its own. But Xu and Goddard calculated that if the reaction started with at least two water molecules, one of them would act as a catalyst driving the reaction forward. It decreases the activation energy barrier from 64.7 kcal/mol to 31.2 kcal/mol if two water molecules were involved; and to 12.0 kcal/mol if 3 water molecules were involved. The reverse reactions have energy barriers of 19.2 kcal/mol and 0 kcal/mol respectively, which suggest that H₂O₃ is not stable in bulk water. The proposed reactions would go as follows: the singlet oxygen reacts with water to form H₂O₃ and a dimer of H₂O₃ then rapidly rearranges to give H₂O₂ and oxygen.

H₂O + ¹O₂ -> H₂O₃                                                 (2)

2 H₂O₃ ->  2 H₂O₂ + ³O₂                                                    (3)

But getting that precise arrangement of water molecules and singlet oxygen to come together is not easy. Perhaps the antibody has a role? To find out, Wentworth and Lerner turned to Ian Wilson, an X-ray crystallographer at Scripps. Using high resolution X-ray Wilson made electron-density maps of the atomic structure of four different antibodies [5]. Scrutinising the regions that all antibodies share in common, Wilson’s group identified three sites close together that could bind oxygen molecules as well as neighbouring sites capable of holding water molecules in the right places.

Isotopic labelling confirmed that the antibodies play a catalytic role in converting ¹O₂ to H₂O₂.

These results were confirmed by another research team at California Institute of Technology, Pasadena [6] using quantum mechanical calculations. The proposed reaction intermediates, HOOOH (H₂O₃) and its dimer are stabilized at the ‘Greek key’ interface of the light and heavy chains, unique to antibodies and T cell receptors, and not present in b₂-microglubulin. This was consistent with the experimentally observed lack of HOOH (H₂O₂) production in the latter as opposed to the former. The catalytic site at which these reactions take place have ordered water clusters (dimers and trimers) which enables ¹O₂ to react with multiple water molecules. In contrast, the binding site for the product, H₂O₂ does not contain any ordered water clusters, and is in a buried hydrophobic pocket.

Further studies comparing the UV irradiated and native structure of the antigen-binding fragments revealed specific modifications to amino acid residues tryptophan L163 and glutamine H6 located in the interfacial regions of the previously identified antibody catalytic site [7].

Burning water kills bacteria

Subsequently,  the Scripps Institute group found evidence that in addition to H₂O₂, ozone (O₃) [8] or other trioxygen species [9] are also produced and released by antibodies, which account for the powerful bactericidal properties of all antibodies, regardless of antigen specificity.

These discoveries are all the more surprising because antibodies have long been cast in the role of “semi-combatant” in the immune system’s war against invading microbes [10]; for it is the immune cells such as macrophages that actually kill bacteria by engulfing them in phagocytosis and digesting them in the vacuoles or phagosomes formed within the cell.

Neutrophils, the most abundant white blood cells in the bloodstream, are also the main phagocytes of the immune system. They kill bacteria and fungi, partly by triggering an oxidative burst composed of a set of enzymatic and chemical reactions that result in the formation of ROS. The first step in this cascade, the reduction of two oxygen molecules, is initiated by the enzyme NAD(P)H oxidase, which becomes assembled in the membrane when the cell is activated by antibody-coated bacteria. The oxidase produces peroxide anion (O₂^{* -} )

NAD(P)H  +  2 ³O₂  ->  NAD(P)⁺  + H⁺  + 2O₂^*-                          (4)

The reactivity of O₂^*- with the cell is relatively low. Its function is to generate more powerful ROS. Its key reaction within the phagosome are either to acquire a proton to become a hydroperoxyl radical (HO₂^*), followed by a fast rearrangement (dismutation) into H₂O₂ and ³O₂, with or without the help of superoxide disumutase enzyme. The H₂O₂ is then used to oxidize Cl^- to hypochlorite OCl^-, a reaction catalyzed by myeloperoxidase. Hypochlorite is highly bacteriocidal but is also used to form chloramines, some of which are even more microbicidal than hypochlorite. An alternative fate of H₂O₂ is conversion into a hydroxyl radical (OH^*).

Given that H₂O₂ and HOCl are present at high concentrations within the phagosome, there is the possibility for a chemical production of high levels of singlet oxygen ¹O₂.

H₂O₂  +  HOCL  ->  ¹O₂ +  HCl  + H₂O                      (5)

As antibodies can catalyze the formation of H₂O₂ from ¹O₂ and H₂O via the intermediate of H₂O₃, with the production of ozone, the Scripps researchers looked for evidence that neutrophils might also produce ozone, and they were successful [11]. The scheme they proposed for the generation of ROS in neutrophils is given in Fig. 1.

Figure 1. Reactive oxygen species generated by neutrophils

That is not the end of the story. In 2008, a team of Japanese researchers led by Kouhei Yamashita at Kyoto University found that four amino acids (without antibodies)  – tryptophan, methionine, cysteine and histidine – are also able to catalyze the conversion of singlet oxygen to ozone when exposed in aqueous solution to UVA radiation. Other water soluble amino acids tested did not have this ability [12].

Indeed burning water seems rather easy, it is a way of life. Animals including human beings do it all the time for signalling purposes and to regulate the energetic status of tissues and cells [13] (Living with Oxygen, SiS 43).

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