**2. Discussion**

### **2.1. The role of oxygen and oxidative stress in prokaryotes evolution**

Oxygen produces only about an order of magnitude more power than fermentation; and the difference between aerobic and true anaerobic respiration is somewhat less than that. While this is substantial, it is orders of magnitude less than the difference made by mitochondria, and probably differences in nutrient availability or concentration gradients outweighed any metabolic advantages of oxygen, at least among bacteria. Oxygen hardly wrought a global revolution in prokaryotic physiology. Even in the presence of oxygen, no prokaryote ever came close to evolving the morphological complexity of eukaryotes. In this context, the evolution of aerobic respiration may have made a difference, but the most immediate impact of the rising tide of oxygen was its juxtaposition with the electron-transport chains of bacteria, all of which transfer single electron [13]. The basic problem, which is central to eukaryotic evolution too, is that the rates of photooxidation and electron transfer, being essentially quantum events, differ from the rates of chemical reduction and carbon assimilation. This means that conditions such as high light intensity (which rapidly photo-oxidizes chlorophyll), low temperatures (electron transfers are barely slowed, but chemical reactions are much slower), and iron deficiency (leading to poor respiratory stoichiometry) all cause high ROS leak. Without compensation, then, ROS leak is largely defined by poor growth: by a low demand for ATP and highly reduced respiratory complexes. There are various ways out of this "high-voltage" situation, from mild uncoupling to complete depolarization of the membrane, or the use of alternative oxidases, which pass electrons directly on to oxygen, without coupling to proton translocation. All of them, in effect, short circuit the membrane potential, enabling faster electron flow, less reduced respiratory complexes and lower ROS leak. If this high ROS leak is not brought under control quickly, the caspase enzymes are activated, significantly by the loss of the respiratory carrier cytochrome c, in plants as well as animals, and the cell is eliminated. Much the same problems affect the respiratory chains of non-photosynthetic aerobic bacteria, among them the free-living ancestors of mitochondria, which likewise are capable of controlled cell death using metacaspase enzymes. The later development of apoptosis in metazoans makes use of enzymes that are bacterial in ancestry, notably the caspases, but also the Bcl-2 family and other mitochondrial apoptotic proteins [34]. Controlled cell death offers the advantage of recycling scarce nutrients, and so can be beneficial to the larger grouping, whether an organism, a colony, or selfish genes [13].

for fertility, fecundity, adaptability, fitness, aging, and age-related disease. Setting the apoptotic threshold high, meaning a high tolerance of ROS leak before apoptosis is triggered, enables high fertility and fecundity. However, the offspring is less fit, and more likely to suffer from mitochondrial diseases. They will have lower aerobic capacity. Worst of all, they will leak ROS from their mitochondria at a faster rate, without triggering apoptosis. The outcome is a shorter lifespan, and a greater tendency to oxidative stress and chronic inflammatory conditions linked with aging, such as diabetes, cardiovascular disease, and cancer. In short, there is a trade-off between fertility, fecundity, and adaptability, on the one hand, and aerobic capacity, lifespan, and susceptibility to age-related disease on the other. The trade-off is mediated by sensitivity to oxidative stress [35]. Thus, oxygen introduces a new penalty for failure, controlled cell death,

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that later played a central role in the evolution of true multicellular organisms [13].

cytochrome P450, NAD(P)H quinone oxidoreductase, and heme oxygenase (HO) [27].

Thiol-based redox signaling is the collective name for biochemical pathways that regulate cellular processes by post-translational modification of sulfur moieties in cysteine (Cys) and methionine (Met) residues of proteins. A single Cys residue can form a redox-sensitive site on a protein. Thus, a redox-active disulfide may be introduced into a protein structure by stepwise mutation of two residues in the native sequence to Cys. By extension, evolutionary acquisition of structural disulfides in proteins can potentially occur via transition through a redox-active disulfide state. However, oxidation of a cytosolic molecule, promoting formation of the disulfide-bonded form, will only occur under conditions of oxidative stress [29].

When a single Cys is present in a protein, conjugation of the redox buffer glutathione may induce conformational changes, resulting in a simple redox switch that effects a signaling cascade. In its role as a redox buffer, GSH is conjugated to reactive Cys of endogenous proteins, inducing conformational changes in the substrate proteins, and effecting a signaling cascade

Although ROS first came to the attention of biologists as potentially harmful by-products of aerobic metabolism, it is now recognized that they play important roles as secondary messengers in many intracellular signaling pathways [25]. This is because as hydrogen peroxide is nonpolar, it is able to diffuse through cell and organelle membranes, and hence acts widely as a second messenger in signal transduction pathways [21]. The redox system can modify functions of proteins through regulating their expression, post-translational modifications, and stabilities. Intracellular redox homeostasis regulates the expression of multiple gene-encoded proteins affecting cell death and survival. In response to alterations in oxidative status, the transcription of those genes can be modulated in part through a redox control of transcription factors such as NF-kB, AP-1, Nrf2, and HIF [36]. Upon exposure of cells to oxidative stress, signaling pathways such as protein kinase C, phosphatidylinositol-3 kinase, and MAP kinase, phosphorylate the transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2). After phosphorylation, Nrf2 translocates to the nucleus and binds to the antioxidant response element (ARE) within the promoters of genes encoding antioxidant enzymes and detoxifying enzymes. Key Nrf2 target genes include glutathione peroxidases (GPx), glutathione S-transferase (GST), superoxide dismutase (SOD),

**2.2. Redox signaling**

that evokes biological responses [28].

The point is that the evolution of metazoan cell death that require apoptosis was built on a system that evolved in relatively complex clonal bacteria capable of an apoptotic-style of cell death in response to oxidative stress. Consequently, the single greatest danger is the failure to pass electrons on swiftly down respiratory chains, resulting in highly reduced complexes in an aerobic atmosphere. The way in which these factors played out in the respiratory chains of eukaryotes may have been one of the most significant selective forces in eukaryote evolution [13]. However, there must be an adjustable threshold, above which ROS leak stimulates apoptosis and developmental failure, and below which ROS leak is tolerated (hormesis), or might even be beneficial as a redox signal [35]. A variable apoptotic threshold has profound implications for fertility, fecundity, adaptability, fitness, aging, and age-related disease. Setting the apoptotic threshold high, meaning a high tolerance of ROS leak before apoptosis is triggered, enables high fertility and fecundity. However, the offspring is less fit, and more likely to suffer from mitochondrial diseases. They will have lower aerobic capacity. Worst of all, they will leak ROS from their mitochondria at a faster rate, without triggering apoptosis. The outcome is a shorter lifespan, and a greater tendency to oxidative stress and chronic inflammatory conditions linked with aging, such as diabetes, cardiovascular disease, and cancer. In short, there is a trade-off between fertility, fecundity, and adaptability, on the one hand, and aerobic capacity, lifespan, and susceptibility to age-related disease on the other. The trade-off is mediated by sensitivity to oxidative stress [35]. Thus, oxygen introduces a new penalty for failure, controlled cell death, that later played a central role in the evolution of true multicellular organisms [13].

## **2.2. Redox signaling**

**2. Discussion**

20 Glutathione in Health and Disease

**2.1. The role of oxygen and oxidative stress in prokaryotes evolution**

whether an organism, a colony, or selfish genes [13].

Oxygen produces only about an order of magnitude more power than fermentation; and the difference between aerobic and true anaerobic respiration is somewhat less than that. While this is substantial, it is orders of magnitude less than the difference made by mitochondria, and probably differences in nutrient availability or concentration gradients outweighed any metabolic advantages of oxygen, at least among bacteria. Oxygen hardly wrought a global revolution in prokaryotic physiology. Even in the presence of oxygen, no prokaryote ever came close to evolving the morphological complexity of eukaryotes. In this context, the evolution of aerobic respiration may have made a difference, but the most immediate impact of the rising tide of oxygen was its juxtaposition with the electron-transport chains of bacteria, all of which transfer single electron [13]. The basic problem, which is central to eukaryotic evolution too, is that the rates of photooxidation and electron transfer, being essentially quantum events, differ from the rates of chemical reduction and carbon assimilation. This means that conditions such as high light intensity (which rapidly photo-oxidizes chlorophyll), low temperatures (electron transfers are barely slowed, but chemical reactions are much slower), and iron deficiency (leading to poor respiratory stoichiometry) all cause high ROS leak. Without compensation, then, ROS leak is largely defined by poor growth: by a low demand for ATP and highly reduced respiratory complexes. There are various ways out of this "high-voltage" situation, from mild uncoupling to complete depolarization of the membrane, or the use of alternative oxidases, which pass electrons directly on to oxygen, without coupling to proton translocation. All of them, in effect, short circuit the membrane potential, enabling faster electron flow, less reduced respiratory complexes and lower ROS leak. If this high ROS leak is not brought under control quickly, the caspase enzymes are activated, significantly by the loss of the respiratory carrier cytochrome c, in plants as well as animals, and the cell is eliminated. Much the same problems affect the respiratory chains of non-photosynthetic aerobic bacteria, among them the free-living ancestors of mitochondria, which likewise are capable of controlled cell death using metacaspase enzymes. The later development of apoptosis in metazoans makes use of enzymes that are bacterial in ancestry, notably the caspases, but also the Bcl-2 family and other mitochondrial apoptotic proteins [34]. Controlled cell death offers the advantage of recycling scarce nutrients, and so can be beneficial to the larger grouping,

The point is that the evolution of metazoan cell death that require apoptosis was built on a system that evolved in relatively complex clonal bacteria capable of an apoptotic-style of cell death in response to oxidative stress. Consequently, the single greatest danger is the failure to pass electrons on swiftly down respiratory chains, resulting in highly reduced complexes in an aerobic atmosphere. The way in which these factors played out in the respiratory chains of eukaryotes may have been one of the most significant selective forces in eukaryote evolution [13]. However, there must be an adjustable threshold, above which ROS leak stimulates apoptosis and developmental failure, and below which ROS leak is tolerated (hormesis), or might even be beneficial as a redox signal [35]. A variable apoptotic threshold has profound implications Although ROS first came to the attention of biologists as potentially harmful by-products of aerobic metabolism, it is now recognized that they play important roles as secondary messengers in many intracellular signaling pathways [25]. This is because as hydrogen peroxide is nonpolar, it is able to diffuse through cell and organelle membranes, and hence acts widely as a second messenger in signal transduction pathways [21]. The redox system can modify functions of proteins through regulating their expression, post-translational modifications, and stabilities. Intracellular redox homeostasis regulates the expression of multiple gene-encoded proteins affecting cell death and survival. In response to alterations in oxidative status, the transcription of those genes can be modulated in part through a redox control of transcription factors such as NF-kB, AP-1, Nrf2, and HIF [36]. Upon exposure of cells to oxidative stress, signaling pathways such as protein kinase C, phosphatidylinositol-3 kinase, and MAP kinase, phosphorylate the transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2). After phosphorylation, Nrf2 translocates to the nucleus and binds to the antioxidant response element (ARE) within the promoters of genes encoding antioxidant enzymes and detoxifying enzymes. Key Nrf2 target genes include glutathione peroxidases (GPx), glutathione S-transferase (GST), superoxide dismutase (SOD), cytochrome P450, NAD(P)H quinone oxidoreductase, and heme oxygenase (HO) [27].

Thiol-based redox signaling is the collective name for biochemical pathways that regulate cellular processes by post-translational modification of sulfur moieties in cysteine (Cys) and methionine (Met) residues of proteins. A single Cys residue can form a redox-sensitive site on a protein. Thus, a redox-active disulfide may be introduced into a protein structure by stepwise mutation of two residues in the native sequence to Cys. By extension, evolutionary acquisition of structural disulfides in proteins can potentially occur via transition through a redox-active disulfide state. However, oxidation of a cytosolic molecule, promoting formation of the disulfide-bonded form, will only occur under conditions of oxidative stress [29].

When a single Cys is present in a protein, conjugation of the redox buffer glutathione may induce conformational changes, resulting in a simple redox switch that effects a signaling cascade. In its role as a redox buffer, GSH is conjugated to reactive Cys of endogenous proteins, inducing conformational changes in the substrate proteins, and effecting a signaling cascade that evokes biological responses [28].

A single Cys residue can form a potential redox-sensitive site on a protein because a second cysteine can be introduced into the sequence with a disulfide formation and oxidation of the cytosolic molecule. The formation of disulfide bridges between two Cys molecules is important in stabilizing protein conformation; therefore sulfur atoms in Cys are responsible for the major covalent cross-links in protein structures. Disulfide bonds between Cys residues are generally thought to confer extra rigidity and stability to their resident protein, forming a type of proteinaceous spot weld. Conformational changes are generally small, involving protein backbone, and are often accompanied by a local increase in protein disorder [28, 37]. Surface modification of proteins by GSH results in significant disorder of the GSH distal to the covalent bond [37].

**3. Conclusion**

**Acknowledgements**

**Conflict of interest**

**Author details**

Roberto C. Burini<sup>1</sup>

MA, USA

**References**

**299**:697-723

**7**:113-123

1978;**10**:3-18

CAPES (HTK graduate fellowship).

The authors declare no conflict of interest.

\*, Hugo T. Kano<sup>2</sup>

\*Address all correspondence to: burini@fmb.unesp.br

The sulfur cycle participation in life evolution comes from ancient anoxygenic elemental sulfur reduction generating environmental sulfide incorporated as mitochondrial Fe-S for the electron-transport chains. Sulfur compounds that contributed for electron leakage and oxidative stress have counteractions by thiol participation either as antioxidant defensors and/or as redox-modulators or cell functions, influencing life evolution and contemporary diseases.

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23

Special thanks to the Brazilian Research Funding CNPq (RCB researcher fellowship) and

and Yong-Ming Yu3

1 Department of Public Health, Center for Nutrition and Exercise Metabolism, Botucatu

2 Pathology Graduate Program, Sao Paulo State University, Botucatu, São Paulo, Brazil

3 Shriners Burns Hospital, Massachusetts General Hospital Harvard Medical School, Boston,

[1] de C, Raiswell R. The evolution of the sulfur cycle. American Journal of Science. 1999;

[3] Olsen GJ, Woese CR. Ribosomal RNA: A key to phylogeny. The FASEB Journal. 1993;

[4] Whittaker RH, Margulis L.Protist classification and the kingdoms of organisms. Biosystems.

[2] Woese CR. Bacterial evolution. Microbiological Reviews. 1987;**51**:221-271

Medical School, Sao Paulo State University, Botucatu, São Paulo, Brazil

GSH plays important roles in nutrient metabolism and regulation of cellular processes, including cell differentiation, proliferation, and apoptosis [38]. DNA-binding activity of transcription factors often involves critical Cys residues, and the maintenance of these residues in a reduced form, at least in the nuclear compartment, is necessary. The reversible thiolation of proteins is known to regulate several metabolic processes including enzyme activity, signal transduction, and gene expression through redox-sensitive nuclear transcription factors such as AP-1, NF-kB, and p53 protein [39]. GSH is involved in a variety of cell functions such as DNA repair, cell cycle, regulation of cell signaling, and transcription factors, GSH therefore can modulate the genes of cell proliferation, differentiation, and apoptosis. The molecular mechanism of how GSH modulates cell proliferation remains largely speculative. A key mechanism for GSH's role in DNA synthesis relates to the maintenance of reduced glutaredoxin or thioredoxin, which is required for the activity of ribonucleotide reductase, the rate-limiting enzyme in DNA synthesis [32].

Due to different roles of ROS in cell signaling and many human pathological processes, imbalance of GSH is observed in a wide range of pathologies including cancer, neurodegenerative disorders, cystic fibrosis, HIV, and aging [30, 40].

### **2.3. Modular redox switches in life evolution**

It is generally believed that before multiple genome sequences were complete, the increased complexity of organisms correlated with the gene number. Hence, after completion of the first genomes, the small differences in gene number between simple unicellular eukaryotes and mammals forced revision of how complexity is encoded. Additional complexity at the organismal level is likely encoded at the molecular level by noncoding DNA [41]. Hence, increased complexity may also be encoded at the protein level. It is recognized that concatenation of existing domains through gene fusion, also known as protein domain mosaicism, encodes new functions in more complex organisms [42]. Studies on the changing amino acid content of proteins show that domains are also not static structures. Additional complexity added to protein domains in the form of redox and other switches likely increases the signaling capabilities of individual domains. In other words, nature is continually tinkering with these independent folding units: a domain from archaea may not have the same sophisticated set of switches as the homologous domain from a mammalian protein. Thus, two modes of acquisition of increased protein complexity have been demonstrated to date: protein domain mosaicism [42] and acquisition of allosteric control sites. Babu et al. [43] showed that Zn finger allosteric control sites are added to protein sequences via retrotransposons. Both are Cysbased sites and are known to be redox regulated.
