Hydrogen Sulfide and Redox Signaling Péter Nagy Department of Molecular Immunology and Toxicology National Institute of Oncology, Budapest, Hungary MUSC Redox Course Charleston, USA, May 2015 H2S and life Sulfur is essential for all forms of life on Earth. In the very early days the atmosphere is believed to have contained large amounts of hydrogen sulfide and only trace amounts of oxygen. Therefore, sulfide may have served as a major life supporter before the emergence of O2. However, sulfide was also pled guilty in causing life destructions and distinctions on the earth most notable during the Permian period. Volcanic eruptions in Siberia caused a major drop in atmospheric and ocean dissolved oxygen levels. It was estimated that by the end of the Permian period 95% of marine species and 70 % of terrestrial ones had vanished. H2S and life Early studies of sulfide biology were focused on its toxic effects. Many death: • Rotorua, New Zealand • Industry: oil wells or refineries, animal processing plants, pump mills, cress-pools, septic tanks… • Sewer gas already mentioned in 1862 Victor Hugo Jean Valjean Sulfide toxicity is mostly associated with cell respiratory dysfunction via impaired oxidative phosphorylation. Mediated by mitochondrial electron transport chain, more specifically Cytochrome C oxidase, inhibition. Sulfide Production in vivo 3MST 3-Mercapto pyruvate Glutamate 2R-SH R-S-S-R Pyruvate AAT -Ketoglutarate L-Homocysteine Cystathionine Cystathionine L-Cysteine H2S CBS L-Cysteine Lanthionine 2 L-Homocysteine SAM CO, NO, SUMO SUMO CSE H2O Homolanthionine H2S -Ketobutyrate L-Cysteine L-Serine Pyruvate + NH3 NH3 Endogenous sulfide production via cysteine metabolism is catalyzed by at least three different enzymatic systems, the main ones being the two pyridoxal phosphate (PLP) dependent CBS and CSE enzymes and the cooperative actions of aspartate/cysteine aminotransferase (AAT) and 3-mercaptopyruvate sulfurtransferase (3MST). Nagy P, Method Enzymol. 2015. p. 3-29. Sulfide Catabolism Sulfide catabolism mostly occurs in mitochondria via oxidative processes driven primarily by the sulfide quinone reductase (SQR) enzyme. The molecular mechanism of this pathway involves initial reduction of an intramolecular disulfide moiety of SQR to produce an SQR-persulfide intermediate. Subsequently, this persulfide functional group is transferred catalytically on to GSH by TST to produce GSSH, which is used as a substrate by a sulfur dioxygenase to give sulfite. Sulfite is than utilized by either sulfite oxidase (SO) or SQR to give sulfate or thiosulfate, respectively. Sulfide Catabolism Despite the major mechanism of sulfide toxicity being inhibition of mitochondrial respiration via interaction with cytochrome C oxidase (CcO), at low concentrations sulfide can also serve as a stimulator of ATP production. Hydrogen sulfide biology Li L and Moore PK, Trends Pharmacol Sci, 29, 84 2008 Hydrogen sulfide biology Radical scavenging Reduction of Reduction of reactive oxidants disulphide bonds We need to better Secondary S understand the chemistry reactive oxidants H H to answer physiological observations! InhibitionCoordination of enzyme to RoleCys sulfhydrtaionin respiration hemefunction proteins Formation of bioactive products Figure 2. Proposed molecular mechanisms Nagy P andfor Winterbourn the interactions CC., Adv. Mol. Toxicol of, 4H, 1822S-222, with 2010 .biological molecules that could be responsible for its physiological effects. Biological concentrations of sulfide; the signal A common detection problem Ka1 Ka2 - 2- H2S + biomoleculeLH2 biomoleculeLH L-H2S adduct • Practically irreversible reactions with sulfide during its detection (that are designed to obtain adequate specificity) take free sulfide out of the system. • As a result of free sulfide consumption, some of the reversibly bound sulfide complexes will start liberating sulfide to attain equilibrium. • Different sulfide-biomolecule complexes liberate sulfide with different rates, which largely depend on the applied experimental conditions. • Therefore, methods that rely on derivatization of sulfide are likely to measure different sulfide concentrations in the same sample, because they often use different conditions and incubation times. • Methods that contain a sulfide precipitation step or volatilization of H2S are also likely to result in a shift in the free vs. bound sulfide equilibria and overestimate free sulfide levels. Nagy P et. al. BBA Curr. meth. to study ROS spec. issue 1840 (2), 876-891, 2014 Abundance and speciation of sulfide in biology Most commonly mentioned sulfide pools • Free sulfide • Acid-labile sulfide Reflects the methods of detection! • Alkaline labile sulfide • Cysteine bound sulfane sulfur Free biological sulfide levels are small but there are efficient buffer systems with large capacities! Sulfide-biomolecule adducts K K H S + biomolecule a 1 biomoleculea2 -H S adduct 2 LH - 2-2 2 LH L Sulfide concentrations in biological samples What are the physiologically relevant concentrations of sulfide that should be used in model in vitro studies? Is it more realistic to use a bolus of sulfide or rather one of the slow releasing sulfide donors? At this point it is difficult to answer these questions, because it should be system specific in a way that it could either be free sulfide, the fast releasing pool or slow sulfide liberation that triggers the corresponding biological function. Chemistry of sulfide signaling Most widely discussed mechanisms of sulfide signaling 1. Protein sulfhydration 2. Interactions with metal centers 3. Cross-talk with NO 4. Sulfhydration of electrophyles Persulfide formation on thiol proteins „Examples of enzyme activation via persulfide formation: 1) Up to 7 fold greater glycolytic activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was observed when the active site Cys150 was modified to a persulfide. 2) Persulfide formation on Cys38 of the p65 subunit of nuclear factor kappa-light-chain-enhancer of activated B cells (NfκB) activated binding to the co- activator ribosomal protein S3 (RPS3) and triggered anti-apoptotic transcriptional activity. 3) ATP-sensitive potassium channels were activated via persulfide formation-induced inhibition of ATP binding. 4) Persulfide formation on Cys341 of mitogen-activated protein kinase kinase (MEK1) leads to Poly [ADP-ribose] polymerase 1 (PARP-1) activation and DNA damage repair via facilitation of phosphorylated extracellular-signal-regulated kinases ½ (ERK1/2) translocation into the nucleus. 5) Nrf2 was activated via Cys151-persulfide formation-induced activity loss of Keap1 (Yang et al., 2013). Inhibition of enzymatic activities via persulfide formation: 1) The previous examples of Nrf2 or PARP-1 activation represent indirect effects, because they are facilitated via persulfide formation-mediated inactivation of their negative regulators, Keap1 and MEK1, respectively. 2) Polysulfide-induced persulfide generation on the active site Cys124 and/or Cys71 residues efficiently inactivated the phosphatase activity of Phosphatase and tensin homolog (PTEN). 3) Persulfide formation on Cys215 had a similar inactivating effect on Protein-tyrosine phosphatase 1B (PTP1B) as the well-established redox switch of the enzyme via reversible cyclic sulfenamide formation between the Cys215 thiolate and its backbone amide nitrogen.„ Nagy P, Method Enzymol. 2015. p. 3-29. Protein persulfides 0 -2 Cys-S-SH -2 -2 Cys-SH H2S It will never occur in the reaction of sulfide with a Cys thiol! One molar oxidizing equivalent is needed! Potential redox cycles for protein persulfide formation in relation to sulfide-signaling. Phisiological oxidant Protein-Cys-SH H2S H2S Protein-Cys-SH Reductant A Protein-Cys-S-SH C HS-OH Protein-Cys-S-OH Protein-Cys-SH HS-SH H2S B H2S (Protein-Cys-S-)2 Protein-Cys-SH Figure 4. Proposed in vivo H2S cycles via intermediate formation of Persulfideprotein species persulfides can. The be persulfide generatedcan be generatedby A direct by A direct oxidation oxidation of the protein ofCys the protein, B Cysdisulfide, B disulfide exchange exchange or C ordirect C oxidation direct of H2oxidationS. of sulfide. Oxidation of sulfide Sulfide readily engages in 1 and 2 electron redox reactions with most of the biologically important ROS and an oxidant scavenging role was proposed for sulfide in various biological situations However, to have an antioxidant role in vivo, these reactions need to be kinetically favored under biological conditions. NOT ENOUGH TO REACT FAST! Relative concentrations! Unlikely that sulfide will have a protecting role against oxidative stress via directly scavenging of ROS in an in vivo situation. Fast sulfide oxidation reactions result in the formation of bioactive oxidation products! Oxidation of sulfide Sulfur has 16 protons and an electron configuration of 1s2 2s2 2p6 3s2 3p4. It has 6 valence electrons and a vacant 3d orbital, which allows it to exist in a wide range of oxidation states (from -2 to +6). -2 Oxidation Polysulfides Oxysulfur species - 2- HSn (n = 2 - 9) e.g.: thiosulfate (S2O3 ) 2- tetrathionate (S4O6 ), 2- SO3 2- SO4 Linking sulfide oxidation with protein persulfide formation Polysulfides oxidize protein thiols Inactivation of PTEN SH SH PTEN + Oxidant PTEN SH SH Active form Inactive form Lee RS. et. al. Journal of Biological Chemistry, 273, 15366-72, 1998 Polysulfides oxidize protein thiols Inactivation of PTEN Greiner R et. al. Antiox. Redox Signal, 19 (15), 1749-1765, 2013 Polysulfides oxidize protein thiols Inactivation of PTEN is due to