Reactive Oxygen Species,Reactive Nitrogen Species and Redox Signaling

NO # ROS # RNS

1. Chapter-10 Reactive oxygen species, reactive nitrogen species and redox signalling

2. Contents 10.1 : Introduction 10.2 : Nitric oxide 10.3 : Reactive oxygen species : superoxide and hydrogen peroxide 10.4 : Interaction of ROS and RNS 10.5 : Hydrogen Sulfide 10.6 : Redox signalling and molecular mechanisms of hydrogen peroxide signalling.

3. Introduction The cellular production of reactive chemical species, especially those based on the reduced states of molecular oxygen, had been known for many years but it was probably the realization that endothelium-derived relaxing (EDRF) was nitric oxide (NO . ) , and that it had profound physiological effects, that started a new area of research. That is, the role of such chemical in cell signaling. There are basically three groups of such chemicals: Reactive oxygen species , referred to a s ROS or AOS (active oxygen species), which include the superoxide anion (O 2 .- ) and hydrogen peroxide (H 2 O 2 ). Reactive nitrogen species (RNS), which are mainly thought of as the nitric oxide radical NO . .However , this can gain and lose electrons to give the NO - and NO + species too (all of which have different chemical properties). Reactive compounds based on sulfur , which for cell signaling mainly means hydrogen sulfide (H 2 S).

4. 10.2 Nitric Oxide The small gaseous molecule nitric oxide is a significant signalling molecule, which its use not restricted to one particular tissue but having functions in various and diverse locations. Nitric oxide is a free radical commonly written as NO . ( the superscript dot denoting its radical status here). That it contains an unpaired electron in its outer electronic orbital. This leads to its increased reactivity as this is an unfavoured electronic state, and one from which a molecule will be “keen” to escape . Here, the NO . c an gain or lose an electron to form NO + or NO - ( as mentioned ) or can be converted to nitrites or nitrates. The signal is usually turned off by its deactivation, where NO . is converted to nitrates and nitrites by oxygen and water.

5. Figure 10.1 Production of nitric oxide from arginine as catalyzed by nitric oxide synthase(NOS). The reaction involves a non released intermediate shown in green. There are several enzymes that can potentially produce NO . , but in animals the most likely is the enzyme nitric oxide synthase(NOS). Here NO . is formed by oxidation of L-arginine . The guanidine group of arginine oxidized in a process, which uses five electrons, resulting in formation of L- citrulline and nitric oxide through an intermediate step in which hydroxy - arginine is formed. This intermediate remains tightly bound to the enzyme and is not released. The substituted analogues act competitively with the binding of arginine , although long exposure to some of these compounds leads to irreversible inhibition of the enzyme . Therefore, such compounds can be used to asses the role of NOS in NO. production in new species or under new conditions

6. Three different NOS isoforms have been characterized. The neuronal NOS ( nNOS, NOS I ) is predominantly expressed in neurons in brain and peripheral nervous system. Endothelial NOS ( eNOS, NOS III ) is mainly expressed in endothelial cells. Both nNOS and eNOS are constitutively expressed and are inactive in resting cells. The third isoform of the NOS family is the inducible NOS ( iNOS, NOSII ). No iNOS expression is found in most resting cells. Exposure to microbial products, such as lipopolysaccharide (LPS) and ds RNA or pro inflammatory cytokines such as interleukin-1 (IL-1), tumor necrosis factor- α ( TNF- α) and interferon- γ ( IFN- γ) induces the expression of iNOS gene in various inflammatory and tissue cells.

7. Figure10.2 A . The domain structure of isoforms of nitric oxide synthase with the area of homology to cytochrome P450 reductase highlighted(blue). B. The proposed structure of the oxygenase domain of nitric oxide synthase ,obtained using X-ray diffraction. Often large proteins such as NOS can be cleaved into smaller sections for structural studies. In the NOS structure, between the binding regions for FMN and FAD and that for haem binding, is a region used for calmodulin binding , which in some cases confers calcium ion control on the enzyme activity. At the N-terminal end of this particular part of the polypeptide is a trypsin sensitive region. Digestion with trypsin yields two domains one from the N-terminal end of NO. synthase, which can bind to arginine and contains haem, and a second containing the NADPH and flavin binding regions .

8. Reactive oxygen species: • Reactive Oxygen Species (ROS) is a phrase used to describe a number of reactive molecules and free radicals derived from molecular oxygen. • These molecules, produced as by products during the mitochondrial electron transport of aerobic respiration or by oxido reductase enzymes and metal catalyzed oxidation, have the potential to cause a number of deleterious events. • It was originally thought that only phagocytic cells were responsible for ROS production as their part in host cell defense mechanisms. • Recent work has demonstrated that ROS have a role in cell signaling, including; apoptosis; gene expression; and the activation of cell signaling cascades. • It should be noted that ROS can serve as both intra- and intercellular messengers. Types of Reactive Oxygen Species

9. Figure 10.3 A scheme showing how NO might fit into a signalling pathway One of the main cellular targets of NO. is the enzyme guanylyl cyclase - is the enzyme responsible for the production of cGMP ,itself an important signalling molecule . For men with dysfunctional penile responses, the drug Viagra is available .It is misconception that Viagra either releases NO., or causes generation of NO.

10. Reactive oxygen species: superoxide and hydrogen peroxide The oxygen consumption was accompanied by an increase in hexose monophosphate shunt leading to production of NADPH. It is now known that the oxygen taken up by phagocytic cells, such as neutrophils, is used in production of superoxide ions , and as discussed in this section, many more reactive oxygen species (ROS) as a result of further reactions. The production of superoxide, however, involves direct enzymatic reduction of molecular oxygen by a complex situated in the plasma membrane of phagocytic cells: this enzyme complex is known as the NADPH oxidase. The primary product from the NADPH oxidase enzyme is the superoxide ion where the electrons are supplied by intracellular NADPH:   2O 2 +NADPH  2O 2 .- +NADP + +H + superoxide ion The extra electron supplied to molecular oxygen is in an unpaired state, and hence like nitric oxide this ion is classified as a free radical . Again, this electronic state is relatively unstable and the new ion is consequently reasonably reactive.

11. 2. For example, it readily undergoes dismutation with formation of hydrogen peroxide   2O 2 .- +2H +  H 2 O 2 +O 2 hydrogen peroxide This reaction occurs spontaneously at low pH Catalysed by enzyme- superoxide dismutase (SOD). 3. Both superoxide ions and hydrogen peroxide are reactive towards biological materials. Both superoxide and hydrogen peroxide can be removed quickly once their potential signals are no longer needed 4. However the real danger comes when superoxide and hydrogen peroxide react together with formation of hydroxyl radicals: O 2 .- + H 2 O 2  OH . +OH - + O 2 hydroxyl radical Hydroxyl radicals are the most reactive chemicals found in biological systems. Damage can be caused to a cell in the form of oxidation of proteins, oxidation of bases that can lead to DNA strand breakage 5. Superoxide can also react with nitric oxide to produce a very reactive compound, peroxynitrite . NO . + O 2 .-  OONO - peroxynitrite

12. Figure 10.4 A Schematic representation of the NADPH oxidase complex and its activation by translocation of several cytosolic components to the plasma membrane. • Besides the membrane bound flavor-cytochrome, the oxidase also requires the presence of several cytosolic proteins for full activity. • Two polypeptides ( 47kDa , p47-phox and 76kDa , p67-phox ) have been identified as being integral with NADPH oxidase activity. • These proteins are primarily found in the cytosol of resting neutrophils, but on activation are translocated to plasma membrane

13. Hydrogen sulfide (H 2 S), like other molecules (nitric oxide (NO•); various isoforms of NO• synthase catalyze NO• production, and carbon monoxide (CO) — a product of heme metabolism) is the inorganic gas, referred to as a gasotransmitter. H 2 S is a mediator of many physiological and/or pathological processes. Some of these effects are ascribed to the formation of protein persulfides, or protein S- sulfhydration , i.e. conversion of cysteine residues –SH to persulfides –S–SH. H 2 S plays an important role in regulating the nervous system and the cardiovascular system. It regulates apoptosis, the cell cycle and the oxidative stress. H 2 S has cardio protective action, neuromodulation properties and may modulate inflammation process, gastrointestinal function, mitochondrial function and energy metabolism.

14. Figure 10.5 Some of the proposed reactions of protein cysteine residues much of the chemistry appears to proceed through the –SOH( sulfenic acid) intermediate. Note that the reverse reactions are not indicated here, but many, although not all, of these steps are reversible • ROS may act on the proteins directly. • The –SH group can be oxidized to the sulfenic acid group, and further oxidized to sulfinic acid and then to sulfonic acid. • Each form of oxidation may have its own effects on the protein function • If there are two thiol groups being oxidized, and they are in close proximity, once the sulfenic acid groups have been formed this allows a further reaction to create a disulfide bridge

15. Figure 10.6 Formation of the sulfenyl-amide intermediate. While studying the oxidized forms of the enzyme, where it was proposed that the cysteine residue in the active site is oxidized, an interesting cysteine derivative was found, the sulfenyl-amide intermediate. This was formed by a reaction of the sulfur of the cysteine linking to the nitrogen of the serine , which was next in the amino acid chain.

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