The gasotransmitters NO, CO and H₂S, have been considered foryears toxic pollutants in the air, to be carefully avoided. Over the last 25years, however, based on solid experimental evidence obtained in a variety of biomedical contexts, they have rather gained the position of leader molecules in cell signaling. GAST are actively synthesized in our body by specific enzymatic systems, and all of them react with similar targets and metalloproteins. NO, particularly, is characterized by a complex, apparently unique mitochondrial reactivity likely suggesting a peculiar physiological interaction with Complex IV.

Keywords: cell respiration, gasotransmitters, nitric oxide, carbon monoxide, hydrogen sulfide, enzyme inhibition, mitochondria, bioenergetics

ATP, adenosine triphosphate; CcOX, cytochrome c oxidase; cGMP, cyclic guanosine-monophosphate; ΔµH+, mitochondrial H+ electrochemical potential gradient; EDRF, endothelium derived relaxing factor; GAST, gasotransmitters; GSH, reduced glutathione; HaCaT, human keratinocyte (Cell Line); HUVEC, human umbilical vein endothelial cells; Km,O2, michaelis menten constant for O2(The Michaelis constant is an inverse measure of the substrate's affinity for the enzyme, i.e. O2 for CcOX, a small Km indicates a higher affinity.); M1, reaction mechanism 1 of NO reaction with cytochrome c oxidase fully reduced; M2, reaction mechanism 2 of NO reaction with cytochrome c oxidase in turnover; mRNA, mitochondrial ribonucleic acid; OXPHOS, oxidative phosphorylation; PKG, protein kinase G; sGC, soluble guanylate cyclase

About 25years work enabled the biomedical science to show that nitric oxide (NO) is only occasionally a poison. All evidence rather suggests that NO is a molecule crucial for life, sharing this feature with CO and H₂S. These gaseous molecules target cell mitochondria. Mitochondria are the sites where the electron and proton transport takes place with free energy changes allowing ATP synthesis.^1,2 The redox chemistry and translocation of H⁺ are both carried out by the respiratory chain complexes. The terminal electron acceptor of the chain, i.e. the complex IV, cytochrome c oxidase (CcOX) performs the O₂ reduction to water, meanwhile pumping 1H⁺/e^- in the mitochondrial inter-membrane space. In doing so, it contributes to built up and maintenance of the mitochondrial H⁺ electrochemical potential gradient, ΔµH⁺. Most if not all the classical mitochondrial effectors/inhibitors bind to CcOX.³ Mitochondrial O₂ consumption normally occurs in the presence of physiological (small) amounts of highly reactive species, including nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H₂S). Once simply considered as toxic air pollutants, these gases are rather endogenously produced by the cells, being therefore recognized as bio-essential.^4,5 Altogether, they are named gasotransmitters (GAST) which are able to trigger and maintain cell-signaling cascades involved in a variety of physiological and pathological events.^6‒8 GAST contribute, among others to regulation of blood pressure, energy metabolism, neoangiogenesis and vascular inflammation, taking part to neurotransmission, cell death and the host immune response.⁹ All GAST have in common the reactivity towards mitochondrial CcOX, although any gasotransmitter shows its own functional features; these depend on the molecular system(s) targeted and on the reaction mechanism(s) involved.¹⁰

Table 1 summarizes some relevant biochemical information on GAST. At body temperature, nitric oxide (NO), carbon monoxide (CO) and hydrogen sulfide (H₂S), similarly to O₂ are freely permeable to phospholipid membranes. From their specific cellular production sites, GAST promptly diffuse in mitochondria and tissues. At mitochondrial level they all react, particularly though not exclusively, with the respiratory chain Complex IV whose oxygen consumption becomes depressed to some variable extent.⁴ Inhibition of respiration and bioenergetic consequences can be physiological or pathological depending on GAST, on its concentration as well as on the functional state of the target.⁴ Although GAST may act in a concerted manner, the knowledge of their reciprocal cross control mechanisms is still poor. Data have been collected strongly suggesting a synergistic action between the NO and H₂S. It was possible to conclude that the hydrogen sulfide, similarly to nitric oxide causes vasorelaxation¹¹ and more recently the H₂S was shown to optimize the NO→cGMP→sGC→PKG pathway, from which the eNOS function depends.¹²

The peculiar mitochondrial reactivity of NO

NO has been the first GAST to be intensively studied after the amazing discovery that it was the endothelium-derived relaxing factor (EDRF).^13,14 Meanwhile, in the early 90’s the involvement of NO in mitochondrial bioenergetics was shown,^15,16 mostly due to its high reactivity towards CcOX, comparable to that of O₂.^17,18 The reaction with NO leads to inhibition of mitochondrial respiration,¹⁹ with depression of the oxidative phosphorylation (OXPHOS) and ATP production. The degree of inhibition and its bioenergetic consequences may be different depending on the cells/tissues exposed to NO. In the brain, for instance, neurons compared to astrocytes, have been proposed to be characterized by a limited glycolytic metabolism.²⁰ Although controversial,²¹ according to the original finding only astrocytes should be able to compensate for OXPHOS ATP loss with glycolytic ATP.²⁰ Experiments carried out using purified CcOX or mitochondria, as well as intact cells proved that the most severe inhibition of respiration by NO is observed at low O₂, as during hypoxia, and at high concentrations of reduced cytochrome c.^18,22‒25 The comparison between the experimental data allowed one to conclude that the extent of OXPHOS inhibition is bound to:

1. The actual concentration of NO whether low (nM) or high (μM), thus its time persistence in the cell environment,
2. The O₂ concentration in the system
3. The level of electron flux within the respiratory chain, and particularly at the Complex IV site (Table 2).²²

One may predict that under rather extreme metabolic conditions, the cell oxidative phosphorylation becomes severely impaired, and NO may become lethal. This likely occurs when a substantial amount of exogenous NO accidentally pollutes the environment. Alternatively, endogenous NO, could be produced in hypoxic tissues by the NOSs, particularly the iNOS.^26,27 Under these conditions NO likely persists in the cellular environment at concentrations >>μM, i.e. to be either efficiently oxidised to nitrite by CcoX in the mitochondrion, and thereafter disposed/recycled, or to be scavenged, e.g. by reduced glutathione (GSH). Such extreme conditions may occur in vivo only during pathological states, such as inflammatory diseases or sepsis,^26,27 when also the concentration of other reactive oxygen species (ROS), particularly the superoxide ion, O₂^-, rises. It is worth to point out that in the presence of both NO and O₂^- the highly toxic peroxynitrite species, ONOO^-, is formed very rapidly at diffusion limited rate.²⁶ Evidence is growing that over and above its own (high) concentration level, the dark side of NO resides on the production of peroxynitrite. In this view it is the ONOO^- that damages membranes, proteins, enzymes and nucleic acids.²⁸ It is worth recalling that CcOX can be inhibited by NO following two different reaction mechanisms,¹⁸ respectively leading to formation of

1. A stable, CcOX NO bound derivative, mechanism 1(M1):

[a₃²⁺Cu_B⁺] + NO → [a₃²⁺Cu_B⁺NO] (NO bound, derivative)

2. A labile, NO₂^- bound, CcOX-derivative, mechanism 2 (M2):

[a₃³⁺Cu_B²⁺] + NO → [a₃³⁺Cu_B⁺NO⁺] → [a₃³⁺Cu_B²⁺ NO₂^-] (NO₂^-bound, derivative)

When the metabolic cell conditions favour M1, the respiratory chain is severely inhibited,^17‒23 and the cells may rapidly undergo apoptosis and irreversible damage, unless alternative ATP-producing pathways, such as glycolysis, become activated. In addition, when M1 prevails, the nitrosylated CcOX does not degrade NO to nitrite, rather releasing the gas back to the environment at the thermal dissociation rate k'=0.01^s-1 at 37°C.²³

At this point it might be worth to consider that the accumulation of the inhibited NO-bound CcOX is favoured by low O₂ and high reducing substrates. It should be also considered, however, that when in cells and tissues the O₂ tension decreases to μM or less, also the NOSs dependent production of NO may become compromised, owing to the lack of O₂, one of the NOSs substrates. Upon lowering O₂, the inactivation of the NOSs occurs, indeed, consistently with their Km,O₂. Thus, sliding towards anoxia, only the endothelial NOS (eNOS), whose Km,O₂ is about 5-6Μm^26,29 may remain active, ensuring some blood vessel dilation. When tissues run out of O₂, however, alternative NO releasing sources may become active; nitroso glutathione (GS-NO) and other NO-donors may act as NO buffers. Moreover, under anoxic conditions, the presence of metal ions (Fe²⁺ and Cu⁺), protein bound or free in solution, promotes the environmental acidification, and at low pH the reduction of NO₂^- to NO is favoured,³⁰ with positive effects on blood flow and oxygenation.³¹ According to this schematic view, a rapid recovery of tissues oxygenation appears to be a priority that prevails on the CcOX recovery (NO would maintain the enzyme inhibited): as a matter of fact, at this point glycolysis should already have taken place!

When M2 prevails, CcOX oxidizes NO to nitrite, thus contributing to NO degradation and detoxification.^22,23 It is worth to point out that under normal physiological conditions, e.g. when NO is endogenously produced at minute concentrations (nM or less) by the constitutive NO synthesis (eNOS and nNOS), M2 likely prevails and the chemistry, responsible for the physiological NO cell signaling predominates over the highly detrimental severe inhibition of mitochondrial oxidative phosphorylation, apparently bound to M1. The physiological relevance of M2 has been clearly shown by the experiments on keratinocytes-derived cells in culture (HaCaT) that proved useful to shed light on the putative physiological bioenergetic role of NO in living cells.³² The experimental design was based on the use of N-acetyl-5-methoxy tryptamine, melatonin, at hormonal concentrations (≤nM) far below the pharmacological ones (μM÷mM). In those experiments the cells, incubated with nanomolar or sub-nanomolar melatonin and under conditions compatible with a circadian rhythm, showed a significant increase of the expression level of both the neuronal NO synthase-mRNA, and the corresponding encoded protein.^32,33 Almost synchronously, the concentration of the NO oxidation products, NOx, i.e. nitrite (NO₂^-) and nitrate (NO₃^-) was found increased in the cell culture medium.

Meanwhile a significant (~30%) depression of both the mitochondrial membrane potential and the oxidative phosphorylation was observed. Interestingly, the depression of the OXPHOS ATP production was compensated by an increased efficiency of glycolysis, directly measured.³³ The melatonin concentration and the time dependence of the onset of the bioenergetic changes both suggest that the NO-dependent nitrite-releasing M2 involving CcOX might play a physiological role in the circadian melatonin chemistry. It is tempting to speculate that the M2 reaction pathway originally observed using CcOX in solution,²³ and confirmed using purified mitochondria and cells in culture^24,25 might contribute to explain the cell bioenergetic changes induced by different pathophysiological stimuli, under those conditions in which a slowdown of the respiratory chain is observed: these conditions should have in common a transient increase of NO leading to a temporary down regulation of the respiratory chain activity, possibly with glycolytic compensation.

Ateneo's Research Project Funding by Sapienza University of Rome is gratefully acknowledged.

Author declares that there is no conflict of interest.

1. Nicholls DG, Ferguson SJ. Bioenergetics. 4th ed. London: Academic Press; 2013. p. 1‒434.
2. Letts JA, Sazanov LA. Clarifying the supercomplex: the higher‒order organization of the mitochondrial electron transport chain. Nat Struct Mol Biol. 2017;24(10):800‒808.
3. Brunori M, Giuffre A, Sarti P. Cytochrome c oxidase, ligands and electrons. J Inorg Biochem. 2005;99(1):324‒336.
4. Cooper CE, Brown GC. The inhibition of mitochondrial cytochrome oxidase by the gases carbon monoxide, nitric oxide, hydrogen cyanide and hydrogen sulfide: chemical mechanism and physiological significance. J Bioenerg Biomembr. 2008;40(5):533‒539.
5. Sarti P, Elena F, Daniela M, et al. Cytochrome c Oxidase and Nitric Oxide in action: Molecular Mechanisms and Pathophysiological Implications. Biochim Biophys Acta. 2012;1817(4):610‒619.
6. Quijano C, Trujillo M, Castro L, et al. Interplay between oxidant species and energy metabolism. Redox Biol. 2016;8:28‒42.
7. Hartmann C, Nussbaum B, Calzia E, et al. Gaseous Mediators and Mitochondrial Function: The Future of Pharmacologically Induced Suspended Animation? Front Physiol. 2017;8:691.
8. Szabo C, Ransy C, Modis K, et al. Regulation of mitochondrial bioenergetic function by hydrogen sulfide, Part I: Biochemical and physiological mechanisms. Br J Pharmacol. 2014;171(8):2099‒2122.
9. Lee SR, Nilius B, Han J. Gaseous Signaling Molecules in Cardiovascular Function: From Mechanisms to Clinical Translation. Rev Physiol Biochem Pharmacol. 2018;2018:1‒76.
10. Kajimura M, Nakanishi T, Takenouchi T, et al. Gas biology: tiny molecules controlling metabolic systems. Respir Physiol Neurobiol. 2012;184(2):139‒148.
11. Hosoki R, Matsuki N, Kimura H. The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergy with nitric oxide. Biochem Biophys Res Commun. 1997;237(3):527‒531.
12. Szabo C. Hydrogen sulfide, an enhancer of vascular nitric oxide signaling: mechanisms and implications. Am J Physiol Cell Physiol. 2017;312(1):C3‒C15.
13. Ignarro LJ, Buga GM, Wood KS, et al. Endothelium‒derived relaxing factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci. 1987;84(24):9265‒9269.
14. Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium‒derived relaxing factor. Nature. 1987;327(6122):524‒526.
15. Cleeter MW, Cooper JM, Darley‒Usmar VM, et al. Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide: Implications for neurodegenerative diseases. FEBS Lett. 1994;345(1):50‒54.
16. Bolanos JP, Peuchen S, Heales SJ, et al. Nitric oxide‒mediated inhibition of the mitochondrial respiratory chain in cultured astrocytes. J Neurochem. 1994;63(3):910‒916.
17. Torres J, Darley‒Usmar V, Wilson MT. Inhibition of cytochrome c oxidase in turnover by nitric oxide: mechanism and implications for control of respiration. Biochem J. 1995;312(1):169‒173.
18. Sarti P, Giuffre A, Barone MC, et al. Nitric oxide and cytochrome oxidase: reaction mechanisms from the enzyme to the cell. Free Radical Biol Med. 2003;34(5):509‒520.
19. Brown GC. Nitric oxide regulates mitochondrial respiration and cell functions by inhibiting cytochrome oxidase. FEBS Lett.1995;369(2‒3):136‒139.
20. Almeida A, Almeida J, Bolanos JP, et al. Different responses of astrocytes and neurons to nitric oxide: the role of glycolytically generated ATP in astrocyte protection. Proc Natl Acad Sci. 2001;98(26):15294‒15299.
21. Diaz‒Garcia CM, Mongeon R, Lahmann C, et al. Neuronal Stimulation Triggers Neuronal Glycolysis and Not Lactate Uptake. Cell Metab. 2017;26(2):361‒374.
22. Sarti P, Forte E, Giuffre A, et al. The chemical interplay between nitric oxide and mitochondrial cytochrome c oxidase: reactions, effectors and pathophysiology. Int J Cell Biol. 2012;2012:571067.
23. Sarti P, Giuffre A, Forte E, et al. Nitric oxide and cytochrome c oxidase: mechanisms of inhibition and NO degradation. Biochem Biophys Res Commun. 2000;274(1):183‒187.
24. Mastronicola D, Genova ML, Arese M, et al. Control of respiration by nitric oxide in Keilin‒Hartree particles, mitochondria and SH‒SY5Y neuroblastoma cells. Cell Mol Life Sci. 2003;60(8):1752‒1759.
25. Mason MG, Nicholls P, Wilson MT, et al. Nitric oxide inhibition of respiration involves both competitive (heme) and noncompetitive (copper) binding to cytochrome c oxidase. Proc Natl Acad Sci. 2006;103(3):708‒713.
26. Sarti P. Nitric Oxide in Human Health and Disease. USA: John Wiley & Sons Ltd; 2013.
27. Yamamoto T, Katayama I, Nishioka K. Nitric oxide production and inducible nitric oxide synthase expression in systemic sclerosis. J Rheumatol. 1998;25(2):314‒317.
28. Pacher P, Beckman JS, Liaudet L. Nitric oxide and peroxynitrite in health and disease. Physiol Rev. 2007;87(1):315‒424.
29. Hall CN, Garthwaite J. What is the real physiological NO concentration in vivo? Nitric Oxide. 2009;21(2):92‒103.
30. Lundberg JO, Weitzberg E, Gladwin MT. The nitrate nitrite‒nitric oxide pathway in physiology and therapeutics. Nat Rev Drug Discov. 2008;7(2):156‒167.
31. Buerk DG, Liu Y, Zaccheo KA, et al. Nitrite‒Mediated Hypoxic Vasodilation Predicted from Mathematical Modeling and Quantified from in Vivo Studies in Rat Mesentery. Front Physiol. 2017;8:1053.
32. Arese M, Magnifico MC, Mastronicola D, et al. Nanomolar melatonin enhances nNOS expression and controls HaCaTcells bioenergetics. IUBMB Life. 2012;64(3):251‒258.
33. Sarti P, Magnifico MC, Altieri F, et al. New Evidence for cross talk between melatonin and mitochondria mediated by a circadian‒compatible interaction with nitric oxide. Int J Mol Sci. 2013;14(6):11259‒11276.
34. Cleva V, Cecilia G. Subcellular and cellular locations of nitric‒oxide synthase isoforms as determinants of health and disease. Free Radic Biol Med. 2010;49(3):307‒316.
35. Gottlieb Y, Truman M, Cohen LA, et al. Endoplasmic reticulum anchored heme‒oxygenase 1 faces the cytosol. Haematologica. 2012;97(10):1489‒1493.
36. Olson KR, Deleon ER, Gao Y, et al. Thiosulfate: a readily accessible source of hydrogen sulfide in oxygen sensing. Am J Physiol Regul Integr Comp Physiol. 2013;305(6):592‒603.
37. Nagahara N, Koike S, Nirasawa T, et al. Alternative pathway of H2S and polysulfides production from sulfurated cat.‒cysteine of reaction intermediates of 3‒mercaptopyruvate Sulfurtransferase. Biochem Biophys Res Commun. 2018;496(2):648‒653.
38. Moller M, Botti H, Batthyany C, et al. Direct measurement of nitric oxide and oxygen partitioning into liposomes and low density lipoprotein. J Biol Chem. 2005;280(10):8850‒8854.
39. Wise DL, Houghton G. Diffusion coefficients of neon, krypton, xenon, carbon monoxide and nitric oxide in water at 10‒60°C. Chemical Engineering Science. 1968;23(10):1211‒1216.
40. Cuevasanta E, Denicola A, Alvarez B, et al. Solubility and permeation of hydrogen sulfide in lipid membranes. PLoS One. 2012;7(4):e34562.
41. Levitt DG, Levitt MD. Carbon monoxide: a critical quantitative analysis and review of the extent and limitations of its second messenger function. Clin Pharmacol. 2015;7:37‒56.
42. Giuffre A, Barone MC, Mastronicola D, et al. “Reaction of nitric oxide with the turnover intermediates of cytochrome c oxidase: reaction pathway and functional effects.” Biochemistry. 2000;39(50):15446‒15453.
43. Bouillaud F, Blachier F. Mitochondria and sulfide: a very old story of poisoning, feeding, and signaling? Antioxid Redox Signal. 2011;15(2):379‒391.
44. Nicholls P, Marshall DC, Cooper CE, et al. Sulfide inhibition of and metabolism by cytochrome c oxidase. Biochem Soc Trans. 2013;41(5):1312‒1316.