ISIS Report 08/06/09

Water and Fire

Living with Oxygen

Scientists across the disciplines are discovering how oxygen created from water is pivotal for the evolution of life on earth and how it affects every aspect of health, disease and development Dr. Mae-Wan Ho

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Oxygen from water pivotal for life on earth

It was the tiny blue-green bacteria (cyanobacteria) that invented the production of oxygen from water and started the great transformation of the earth’s atmosphere to one suitable for aerobic metabolism and the evolution of complex life-forms some 2.5 billions years ago..

 Photosynthesis as we know it today is responsible not only for the vital task of taking CO₂ out of the atmosphere, but also for replenishing the atmosphere and the oceans with oxygen that’s essential to sustain all aerobic life.

The creation of the photosynthetic apparatus capable of splitting water into oxygen, protons, and electrons, was the pivotal innovation in the evolution of life on Earth. It gave access to water, a new, unlimited source of electrons and protons for photosynthesis [1], thereby greatly expanding the thermodynamic range of energy available to life. Before that, some bacteria carried out a form of photosynthesis that does not produce oxygen and depends on the limited availability of chemical substances that can act as electron donors.

The primitive photosynthetic pigment, bacteriochlorophyll a (BChl-a), found in green non-sulphur bacteria, when excited by sunlight, has a standard electrochemical potential of 0.55 V, which is sufficient for using ferrous iron, carbon and sulphur substrates as electron donors. To use water as electron-donor, and hence to produce oxygen, requires the creation of the chlorophyll-a in cyanobacteria and green plants that can be boosted to a higher electrochemical potential of 0.82 V, thereby giving life access to a higher energy level and a practically unlimited energy source. This event literally changed the face of the earth. The accumulation of O₂ in the atmosphere led to aerobic respiration, a much more efficient transformation of energy that generates 18 times more molecules of ATP (adenosine triphosphate) from one sugar molecule than anaerobic respiration. ATP is the universal intermediate for energy transduction in living organisms, and is therefore a convenient measure of useful energy.  Aerobic metabolism, in turn, enabled the evolution of complex, multicellular, energy-efficient eukaryotic organisms, as Lauren Koch and Steven Britton at the University of Michigan Ann Arbor in the United States argue in a comprehensive review published in the Journal of Physiology [2].

To appreciate the momentous importance of getting oxygen from water, one has to realise that life is energized by nothing more than the intricately orchestrated flows of electrons and protons carrying negative and positive electricity respectively. In the process, life creates itself and transforms the entire earth [3] (see The Rainbow and the Worm, The Physics of Organisms, ISIS publication).

Oxygen and the evolution of complex life forms

Life on earth is generally believed to have originated 3.9 billion years ago, though some scientists have recently put it back as far as 4.4 billions years ago [4]. The atmosphere was devoid of oxygen, and hence the primordial organisms were solely dependent on getting electrons from organic compounds like sugar or inorganic molecules such as hydrogen, sulphide/sulphur, ammonia, metal/metal ions. Anaerobic respiration cannot make much energy available to support many activities. Consequently, no multicellular complex organisms are exclusively anaerobic.

Bacteria capable of harvesting light energy in a primitive photosynthetic process that does not produce O₂ appeared about 3.3 billions years ago. Oxygen-producing photosynthesis only became established around 2.5 billion years ago. That initiated the Great Oxidation Event to build up O₂ in the atmosphere to 2 percent by 2 billions year ago. The O₂ level remained static for the next billion years when aerobic respiration evolved and small multicellular organisms became widespread. Between 1.0 and 0.5 billion years ago, O₂ rose sharply to its current level of 20 percent. This increase is widely believed to have fuelled the ‘Cambrian explosion’, the rapid evolution of all major animal phyla on earth between 540 and 490 million years ago [5]. Oxygen level then took a steep dive to below 15 percent before rising sharply to 35 percent 300 million years ago, coincident with the evolution of large woody land plants with very active photosynthesis [6]. This period saw the emergence of gigantic insects, as the high atmospheric O₂ concentration lifted the constraint on its availability to tissues and cells supplied through the insect’s tracheal system [2]. The subsequent drop in O₂ concentration to more or less the present 20 percent  ~260 to 234 million years ago is believed to be due to a substantial reduction in lowland forests and swamps.

A rough correlation is found between increasing oxygen levels and the rise in the number of different cell types estimated from the protein sequence data using molecular clock methods [7] (see Development and Evolution Revisited, ISIS scientific preprint).. Organisms with two to three cell types appeared shortly after the initial increase in atmospheric O₂ to 2 percent 2 billions years ago, with further increases occurring only when O₂ started to rise again at 1 billion years ago up to 120 cell types by 0.5 billion years ago when oxygen levels reached 20 percent..

Oxygen is stable, abundant, and fit for life

It is of interest that life is created out of the 85 stable elements; and 7 of the most abundant 10 elements in the universe - hydrogen, helium, oxygen, carbon, neon, nitrogen, magnesium, silicon, sulphur and iron – are represented in organisms. So, it is very probable that life anywhere else in the universe would also be similar to that on earth.

Oxygen is the third most abundant element and has special features that make it most fit for life. First, the molecules of life are carbon-based, and among the elements, only nine are more electronegative than carbon and therefore able to serve as an acceptor of electrons from carbon-based substrates. Of the nine – selenium, sulphur, iodine, krypton, bromine, nitrogen, chlorine, oxygen, fluorine – oxygen ranks second only to fluorine in electronegativity, and the other elements are either a solid, highly reactive, less abundant, or substantially less electronegative. Thus, reduction of oxygen (burning, transferring electron to oxygen) provides close to the largest possible transfer of energy for each electron. Another advantage of oxygen is that it exists typically as a stable ‘triplet’ molecule consisting of two atoms (O₂) [8] (see The Body Does Burn Water, SiS 43), which is a freely diffusible gas, and hence available to all life forms, in the air, on land and in water. Furthermore, O₂ gas is much more easily transported to tissues and cells inside the organism.

Oxygen in action past and present

Large evolutionary events are associated with oxygen in the past (see above). But the evidence is also all around us today [2].

For example, the trend for animals to be larger at higher latitudes – polar gigantism - .is best correlated with oxygen. A measurement of 1 853 bottom-dwelling amphipod crustaceans from 12 sites worldwide that included polar, tropical, marine and freshwater environments revealed a strong correlation between maximum body length and oxygen content (r² = 0.98, p<0.0001). In the lab, a reduction of O₂ level by 10 percent decreases body mass of fruit flies, while an increase O₂ level of 40 percent increases body mass. The atmospheric oxygen burst due to land plants that boosted O₂ level in the earth’s atmosphere to 35 percent 300 million years ago and its subsequent decline to present levels was indeed accompanied by a dramatic rise and fall in the size of insects.

The fossil record shows that vertebrates started to come on land about 415 million years ago. Thereafter, the record disappeared for the interval 360-345 million years ago. This 15 million years lull in the fossil record of vertebrates’ colonization of land, known as Romer’s Gap, remained a complete mystery until a research team tested the hypothesis that environmental factors might be responsible. They looked at the ranges of terrestrial arthropods over the same time period, and found a pattern similar to that of vertebrates; that is, few new groups evolved for both vertebrates and arthropods during Romer’s Gap. They suggested that it coincided with, and is explained by the low atmospheric O₂ at the time, less than 15 percent at its lowest

Current global warming is particularly challenging for fish because warming of the oceans results in less dissolved oxygen at the same time that warm temperatures pushes up metabolic rate. A study of eel pouts from the North and Baltic Seas revealed that thermally limited oxygen delivery closely matches environmental temperatures, and there is a threshold beyond which growth and abundance decrease. Consequently, warming seas will be the first process to cause extinction or relocation of organisms to coolers water.

Actually, warming seas hit photosynthetic plankton first, and has already done so. Plankton is the major food source that supports marine life as well as replenishes oxygen in seawater [9, 10] (Shutting Down the Oceans. Act II: Abrupt Plankton Shifts, and Shutting Down the Oceans Act III: Global Warming and Plankton; Snuffing Out the Green Fuse, SiS 31). The amount of plankton biomass created is the difference between photosynthetic rate and respiration rate. As temperature rises, both photosynthetic and respiration rates go up. Unfortunately, respiration goes up faster, resulting in less plankton growth.

Oxygen and complexity of the metabolic net

Oxygen makes more energy available and at greater efficiency; at the same time, it increases the complexity of metabolic networks.

The core metabolic network connects major chemical species (metabolites) that are transformed by biochemical reactions involved in energy transduction and mobilisation. It is a very complicated branching network that offers infinite possibilities for channelling and diverting metabolites to different ends for maximum effect [11].

The connectivity of the network of 43 different organisms in all three domains of life from bacteria to higher organisms was investigated using the tools of graph theory and statistical mechanics [12]. The analysis revealed that the metabolites are not randomly connected. Instead the number of connections per node (metabolite) approximates a power law, P(c) ~ c^-g, where c is the number of connections, and P(c) is the probability of finding a node with c connections. In other words, most metabolites have one or a few connections, and the number of nodes with many connections drops off rapidly. Significantly, the most highly connected metabolites were those associated directly with energy transfer, with water at the top of the list!

This ‘scale-free’ or fractal network has been found to describe biological structures and living processes in general [13, 14] (Biology’s Theory of Everything, SiS 21; The Heartbeat of Health, SiS 35). It also applies to the World Wide Web, Internet, and social networks [12].

A direct investigation on how oxygen availability changed the architecture of metabolic networks was carried out by ‘seeding’ with a pre-specified set of metabolites that were allowed to react according to known enzymatic reaction rules. Once all possible reactions have been carried out, the products then join the seed metabolites, potentially allowing new reactions to occur. This process was reiterated until no new products were generated [15]. The analysis separated four discrete groups of networks of increasing complexity, with transitions between groups contingent on the presence of the key metabolites: NAD⁺, S-adenosyl methionine, coenzyme A, ATP O₂, CO₂, NH₃, pyruvate or 2-oxoglutarate. The most complex group IV reactions were associated almost exclusively with the presence of O₂, and had as many as 1 000 reactions more than those of the largest networks achieved without O₂.

Oxygen in health and disease

Clinical studies reveal a strong statistical association between low aerobic capacity and death from all causes. In other words, failures of oxygen and energy metabolism underlie all complex diseases.

In one study, researchers followed 6 213 men for 6.2 years, who were referred for treadmill exercise testing for clinical reasons. The subjects were classified into two groups: 3 679 had an abnormal exercise-test result and/or a history of cardiovascular disease, and 2 534 had a normal exercise-test result and no history of cardiovascular disease. In both groups, the peak exercise capacity was a stronger predictor of an increased risk of death compared with established risk factors such as hypertension, smoking, diabetes, or the development of arrhythmias during exercise. In all subgroups, the risk of death from any cause in subjects whose exercise capacity was less than 5 MET (metabolic equivalents) was roughly double those of subjects whose exercise capacity was more than 8 MET. Each 1 MET increase in aerobic exercise capacity was associated with a 12 percent increase in survival.

Numerous studies also linked declines in aerobic capacity and mitochondrial function with type 2 diabetes, cardiac arrhythmias, inflammatory response, longevity and aging. Physical exercise and aerobic capacity are associated with reduced cancer risk. Intriguingly, cancer cells are characterized by a reversion to anaerobic respiration or glycolysis.

Since about 20 years ago, Koch and Britton have tried to create an animal model that reflects the polygenic nature of a complex disease such as type 2 diabetes or hypertension.  They have come to reject every known approach including chemical and physical manoeuvres to mimic diabetes, single or multiple gene knockout models, and mutagenesis. So in 1996 they embarked on large-scale selective breeding to develop strains of rats that contrast for intrinsic aerobic treadmill running capacity using a founder population of 96 breeding pairs [2]. After 21 generations of selection, the low-capacity runners (LCR) and high capacity runners (HCR) differed by 461 percent in aerobic running capacity.

The LCR scored higher as young adults on cardiovascular risks and features of the metabolic syndrome of diabetes, including higher blood pressure, insulin, random and fasting glucose levels, free fatty acids, visceral adiposity and triglycerides. The HCR in contrast, measured higher for health factors such as maximum oxygen uptake, heart function, endothelial nitric oxide formation, economy of oxygen use and levels of transcription factors and oxidative enzymes. When exposed to high fat diet, LCR rats gained more weight and fat mass, and became more insulin resistant. Remarkably, the same metabolic variables remained unaltered in HCR rats shifted to the high fat diet.

Reactive oxygen species in health and disease

While oxygen has been incorporated into aerobic metabolic processes and became essential for life, it also produced reactive by-products that damage the molecules required for life. Consequently, organisms have evolved mechanisms to protect themselves from oxidative damage and to repair the damage caused by oxygen.

Reactive oxygen species (ROS) refers to a variety of molecules and free radicals derived from molecular oxygen [8]. They are unavoidable by-products of aerobic respiration and various other catabolic and anabolic processes. The respiratory chain produces superoxide that can be converted to peroxide by superoxide dismutases. Enzymes that produce ROS as by-products include fatty acyl-CoA oxidase, xanthine oxidase, cytochrome p450 systems, cyclooxygenases and lipoxygenases. ROS are directly produced from oxygen by NADPH oxidases, a major family of enzymes first detected during phgocytosis in special white blood cells, an engulfing process whereby invading microbes are destroyed; but are now known to be broadly distributed in many tissues, and increasingly implicated in signal transduction processes and intercommunication [15]. Specific trans-membrane proteins, aquaporins, initially described as water channels present in all cells, facilitate the ROS hydrogen peroxide to diffuse across cell membranes.

Cells have many ways to respond against ROS using enzymatic and non-enzymatic antioxidants. Common non-enzymatic antioxidants are glutathione (GSH) and thioredoxin. Glutathione synthesis is catalyzed by the sequential action of g-glutamylcysteins synthetase and GSH synthetase. GSH is oxidized to GSSG, and the reduce form can be regenerated by GSH reductase. The balance between GSH and GSSG determines the redox (reduction-oxidation)/energetic state within the cell. Thioredoxins (Trx) as well as glutaredoxins (Grx) are small proteins containing an active site with a redox-active disulfide, and are involved in maintaining a reduced intracellular redox state in mammalian cells by the reduction of protein thiol (SH) groups.

Antioxidant enzymes act in concert to remove various ROS produced. Superoxide dismutases scavenge the superoxide radical, converting it into hydrogen peroxide and oxygen. Catalase and the peroxidases convert hydrogen peroxide into water and oxygen. GSH and Trx are also specific substrates for a group of peroxidases. The glutathione peroxidases are a group of selenoproteins that catalyze the reduction of peroxides generated by ROS at the expense of GSH. Four have been identified in mammals. The Trx-dependent peroxidases are known as peroxiredoxins and six have been identified in mammals.

An oxidative stress is generated when a cell accumulates an excessive concentration of ROS beyond what the antioxidant defence can cope with. As a passive effect ROS can be detrimental to cells due to oxidative damage to lipids, proteins and DNA. Among the products are lipid hydroperoxides, carbonylated proteins and DNA with oxidized bases.

While ROS defence is well-established, it is now also generally accepted that ROS are signalling molecules that transduce messages from the extracellular milieu to cells to generate a specific response. ROS have defined functions through modifications of a great diversity of molecules participating in almost every signalling pathway in the organism. In some cases redox regulation is indirect through interaction with Trx, for example.

Proteins undergo redox modifications mainly at cysteine (Cys) residues although other amino acids can be oxidized, such as Tyr, Trp, His. Proteins subject to ROS modification include kinases and phosphatases that are themselves key regulators of cellular functions through chemical modifications of yet other proteins.

Transcription factors that regulate transcription of DNA, likewise, are subject to ROS regulation. ROS modifications can activate or inactivate the target proteins

ROS is involved in signalling pathways of growth factors and cell proliferation, and many studies suggest that they may function as classical second messenger in that the specific response depends on the cell type, the intracellular compartment where the ROS is produced, the specific ROS and dose. For example, low and high hydrogen peroxide concentrations produce a contrasting effect on cells that is dependent on the apoptosis protein p-53; low doses cause anti-oxidant mediated survival, whereas high doses promote pro-oxidant mediated cell death.

ROS is now implicated in all aspects of development, from germ cell formation to morphogenesis, formation of blood vessels and cell migration, reviving interest in the century-old hypothesis that metabolic gradients guided early development, and other mysteries of morphogenesis.

Finally, ROS produced by the immune system as defence against invading microbes, and the extraordinary ability of antibodies and other proteins to produce powerful bacteriocidal ROS are described in another article of this series [8].

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