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 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) . 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 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 interactionsCC., Adv. Mol. Toxicol of, 4 H, 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

Ka1 Ka2 - 2- H2S + biomoleculeLH2 biomoleculeLH L-H2S adduct

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 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 persulfide and potentially ciclyc trisulfide (for WT) formation at the active site Cys residues

Polysulfides oxidize protein thiols

Inactivation of PTEN inside the cell

Is it polysulfides that form in the medium and transported into the cell or sulfide oxidation inside the cell? Making and handling sulfide solutions

Measured concentrations for a 150 mM stock solution Sulfhydration via Cys oxidation

R-SH + H2O2 R-SOH + H2O

1. In general CySOH is a reactive short lived oxidative Cys modification.

2. We established estimated the rate constant for its reaction with thiols to give disulfides to be > 105 M-1s-1. Expected to vary in proteins.

R-SOH + H2S R-SSH + H2O

P. Nagy, and MT. Ashby, JACS., 129, 14082, 2007. Modeling cellular thiols as targets for H2O2

• Selectivity will depend on the reactivity of the Cys residues with

ROS as well as on the CySOH with H2S. Persulfide formation via disulfide reduction

R-SSR’ + H2S R-SSH + R’-SH

1. It was suggested that sulfide is a not very powerful reducing agent

2. Thermodynamics vs kinetics

Francoleon NE, ABB, 516, 146-153, 2011. Nagy P. Antioxid Redox Signal, 18 (13), 1623-1641, 2013 Disulfide reduction by sulfide

Vasas A et. al. Nitric Oxide, 2015 46, 93-101. Disulfide reduction by sulfide

1. Reduction of disulfides by sulfide is feasible under physiological conditions both on thermodynamic and kinetic grounds.

2. The reaction occurs via multistep equilibria along the disulfide persulfide inorganic polysulfides axis.

3. Just as for Cys-thiol-disulfide exchange reactions  protein specific speciation

CBS and CSE use cystine to produce persulfides

Ida T et. al, PNAS. 2015. 111:21. 7606. Detection of Cys Sulfhydration

Method 3

By Solomon Snyder, Kate S. Carroll and Milis Filipovic

Interaction of sulfide with metal centers

Reactivity and mechanism depends on:

1. Accessibility of the active site

2. Nature and abundance of different enzyme forms (pl. Comp.I or Comp.II)

3. H2S concentration

4. Properties of the neighboring functional groups • Polarity of the active site environment • Close proximity of H-bond donor or acceptor functional groups

.- H2S HS Ka1 Ka2 FeIII - III 2- II + H2LSH 2 LH Fe L Fe

Low spin complex

Pietri R et. al. Antiox. Redox Signal, 15(2), 393-404, 2011 Interaction of sulfide with

Interaction of H2S with myeloperoxidase Palinkas Z et. al. Br J Pharmacol, 2015 172, 1516-32 Interaction of sulfide with myeloperoxidase

Low-spin sulfide bound Fe(III)

High-spin Fe(III)

Sulfide binds to the MPO active site Interaction of sulfide with myeloperoxidase

K K H2aS1 a2 H2S - 2- MPOFeL(III)H2 LMPOFeH (III)-HL2S complex MPOFe(II)-H2S complex Ferric form

H2S

MPOFe(II) Ferrous form

Sulfide also reduces the Fe(III) center to Fe(II) Sulfide is a very efficient inhibitor of MPO activity

Sulfide-mediated MPO inhibition is fully reversible

Inhibition of MPO in biological samples

rat neutrophil lysates

rat inflamed colon homogenates

human neutrophil lysates

IC50 value of ~ 1 µM as with the purified enzyme Proposed model for the interactions of sulfide with MPO

Polysulfides Cross talk between redox and sulfide signaling

Interaction of sulfide with heme mediate the chemical nature and kinetics of ROS production.

Therefore, this in light of the well documented redox regulation of cellular signaling could represent an alternative element of sulfide’s regulatory role.

MPO and its oxidants can orchestrate the relative amounts of reduced vs oxidized sulfide forms (that will have different biological functions)

MPO inhibition is via coordination chemical interaction of sulfide rather than its oxidation products

Could serve as a prime example for a fine tuned signaling system!

Heme protein interactions with sulfide

Cross-talk between sulfide and NO signaling

Kolluru GK et. al. Methods Enzymol, 2015 554, 271-97 Kolluru GK et. al. Redox Biol, 2013 1, 313-8 Cross-talk between sulfide and NO signaling

Sulfide mediated sustained NO

Transnitrosation via HSNO bioactivity of nitrosothiols

Filipovic, M. R, et. al. JACS,, 134, MM Cortese-Krott MM. et. al. Redox 12016-12027, 2012 Biology, 2,234–244, 2014

Marian Grman Electrophile sulfhydration

H2S can sulfhydrate electrophilic messengers. Electrophiles are generated as byproducts of lipid peroxidation with widespread regulatory roles. Sulfide could modulate their actions.

Nishida M, et al. Nat. Chem. Biol. 2012 714-724. Kabil O, et al. ABB 2014 Redox biochemistry of Cys

Physiological roles of cysteine oxidation

1) Oxidation to disulfide bonds is essential for the correct folding of newly synthesized polypeptides.

2) Reduction of deleterious oxidants (antioxidant character)

3) Dynamic redox reactions of Cys residues govern the actions of and enzymes.

4) A wide array of proteins are regulated via redox reactions of their active site or regulatory Cys residues, including transcription factors, metabolic enzymes, membrane channels and proteins in phosphorylation signaling pathways. Redox biology

The defining lines between oxidative stress-mediated cellular damage, redox sensing and redox-based signaling events need to be better understood.

Historically, focus was on the antioxidant properties of Cys-residues, primarily in glutathione, to alleviate oxidative stress-induced damage, with subsequent work establishing redox sensing as a key component of cellular adaptation to stress signals.

Today, thiol based redox events are considered essential for most aspects of normal cellular physiology, including enzyme , protein structure, and as intramolecular switches. Compartmentalization

Different redox processes may operate independently in different cellular compartments.

-Protein folding is mostly carried out by dedicated oxidation machineries of the endoplasmic reticulum (ER), mitochondrial intermembrane space (IMS) of eukaryotic cells or in the periplasm of prokaryotes.

-Efficient antioxidant systems operate in the mitochondrial matrix, which hosts oxidative phosphorylation and core metabolic machineries that generate superoxide and H2O2 as by-products.

-Most of the known thiol based signaling pathways exist between the cytosol and the nucleus to govern normal cellular functions and pathologic conditions.

All these processes need to be studied in a compartment specific manner.

CO H CO - 2 Thiol pKa is an 2important parameter that determines reactivity+ HC CH2SH HC CH2SH + H pKa1 = 1.71

+ + NH3 NH3 - - CO2 CO2

- + HC RC-HSH2SH + HOx H C CH2S + Products H pKa2 = 8.slow33

NH + + 3R-S- + HOx N H 3 Products fast - - CO2 CO2 [RS-][ H+] - - - -+ + + RSH HC RC-HSH2S RSH RH C - S R C+SH 2H+S H + H K apKa3 == 10.78 (15) [ RSH] + NH3 NH2 RSH + R'SSR'' i RSSR' + R''SH (16)

RS- + R'SSR'' RSSR' + R''S- (17) Acid-base equilibria for cysteine

- CO2H CO2

+ HC CH2SH HC CH2SH + H pKa1 = 1.71

+ + NH3 NH3 - - CO2 CO2

- + HC CH2SH HC CH2S + H pKa2 = 8.33

+ + NH3 NH3 - - CO2 CO2

- - + HC CH2S HC CH2S + H pKa3 = 10.78

+ i NH3 NH2

HA. Sober, Ed., “Handbook of Biochemistry” 2nd ed; Cleveland OH, 1970 Acid-base equilibria for cysteine

- CO2H CO2

+ HC CH2SH HC CH2SH + H pKa1 = 1.71

+ + NH3 NH3 - - CO2 CO2

+ HC CH2SH HC CH2SH + H pKa2 = 8.7

+ NH3 NH2 - - CO2 CO2

HC CH SH HC CH S- + H+ pK = 10.8 2 i 2 a3 NH2 NH2 DD Perrin, “Dissociation Constants of Organic Bases in Aqueous Solution” Butterwoths: London, 1965 Acid-base equilibria for cysteine

- - CO2 CO2

- + CH CH2SH CH CH2S + H pKa2 = 8.65

N(CH3)2 N(CH3)2

- - CO2 CO2

+ CH CH2S-CH3 CH CH2S-CH3 + H pKa3 = 8.75

+ NH3 NH2

GM Bodner, J. Chem. Educ. 63, 246, 1986 Scheme 3. Protonation AcidStates for- CybaseSH. equilibria forAc idcysteine/Base Equilibri a for CySH. CO2H CySH+ + HC CH2SH = CySH CySH + NH3 Ka1 - CO2 CySH0 0 HC CH2SH = CySH CySH CySH Ka2n Ka2s + NH3 CySH - Kex CO2 CySH- CyS- - HC CH2SH = CySH CySH CySH K K NH2 a3s a3n 2- - CyS CO2

- - HC CH2S = CyS {[CyS- ][CySH- ]}[H ] NH + CySH CySH 3 Ka2  Ka2s  Ka2n  0 - [CySH ] CO2 KCySH KCySH [CyS2- ] [H ] HC CH S- = CyS2- a3s a3n 2 Ka3  CySH CySH  - - Ka3s  Ka3n [CyS ][CySH ] NH2

D. Garfinkel and JT. Edsall, JACS. 80, 3823, 1958 Protein thiol pKa

- Neighboring charged, aromatic or H-bond donor or acceptor functional

groups will have a pronounced effect on the thiol pKa.

-All experimental methods to measure pKa values include pH titration  proteins undergo conformational changes as the pH is varied.

-It is therefore hard (if not impossible) to evaluate the contribution of the protonation/deprotonation of one particular functional group in a pH titration dataset of a protein.

-In most proteins there are more than one Cys residue and in many cases

spectrophotometric and kinetic pKa measurements cannot differentiate between them.

-Changes in spectroscopic properties are usually not specific to one type of functional group.

- Kinetic method is the best. Accessibility to the active site can affect kinetic measurements by alkylating agents. Use the physiological substrate of the protein of interest.

Nagy P and Winterbourn CC., Adv. Mol. Toxicol, 4, 182-222, 2010. - CO2H CO2

+ HC CH2SH HC CH2SH + H pKa1 = 1.71

+ + NH3 Thiol pKa is anNH 3important parameter that - - CO2 determinesCO2 reactivity

- + HC RC-HSH2SH + HOx H C CH2S + Products H pKa2 = 8.slow33

NH + + 3R-S- + HOx N H 3 Products fast - - CO2 CO2 [RS-][ H+] - - - -+ + + RSH HC RC-HSH2S RSH RH C - S R C+SH 2H+S H + H K apKa3 == 10.78 (15) [ RSH] + NH3 NH2 RSH + R'SSR'' RSSR' + R''SH (16) The nucleophilicity of the thiolate increases with thiol pKa!

- - (17) logk = -1.29 + RβS pK+ RRSH'SS R'' RSSR' + R''S nuc a i Influence of thiol pKa on the rate of its nucleophilic reactions at pH ~ 7

100

) -1

s 80 -1

(M 60

app

1 k 40

20 Calculated Calculated 0 4 5 6 7 8 9 10 Thiol pK a

Decreasing the thiol’s pKa will only result in an overall rate enhancement until the thiolate becomes ithe dominant species (i.e. until the thiol pKa approaches the applied pH).

Nagy P. Antioxid Redox Signal, 2013 18, 1623-41. The pKa of the oxidant is also an important rate determining factor

R-SH + HOx Products

6 )

-1 5 s

-1 4 (M

-7 3

2

x x 10 eff

k 1 0 80

60 pK TNB-SHo pK HOSCN 40 a a TNB-SH-

Percentage pK 20 a 0 i 2 3 4 5 6 7 8 pH

Nagy P. et. Al. Chem. Res. Toxicol. 22, 1833, 2009. In order to oxidize Cys you need a reaction partner i.e. an oxidant (even in biological systems)

The popular term Reactive Oxigen Species (ROS) does not mean much.

Different oxidants have very different chemical properties!

YOU NEED TO (better) KNOW YOUR OXIDANT!

Hypohalites

Hypochlorite Hypobromite Hypothiocyanite - - OCl OBr

• Relatively stable at • Less stable at • Unstable at neutral neutral pH neutral pH pH

• pKa = 7.4 • pKa = 8.6 • pKa = 4.8

• HOCl is a powerful • HOBr is a better • HOSCN is a poor oxidant oxidant oxidant

• Reacts with RSH, • Reacts with RSH, • Reacts with RSH only

RSR, R2NH, RSR, R2NH, olefins, olefins, aromatics, aromatics, etc.i etc. Neutrophils generate reactive oxidants

1 e P a t h w a y s 2 e P a t h w a y s I N T R A C E L L U L A R E X T R A C E L L U L A R ( O R P H A G O S O M A L )

F e 3 + / F e 2 + O H N A D P H O 2 N A D P H o x i d a s e N A D P + + H + - O 2 H 2 O 2 H 2 O S O D N O S N O Br- M P O HOBr H 2 O H 2 O 2 R H S C N - C l -

R O S C N - O N O O - H O C l

H O - 2 2 O 2

R N H 2 1 O 2 O H R N H C l

MB. Hampton et al. Blood. 92, 3007, 1998 2 e- redox reactions of cysteine R-SH Ox

R-S-Cl R-S-Br R-S-SCN R-S-OH Ox RSOH RSH R'R''NH HX HX HX PTP OH RSSR H2O Ox R'R''NH RSH R' O R-S R-S-S-R R-S-N O R-S-S-R R'' Ox Ox RS(=O)SR Ox RSSR H O RSSR RSSR 2 RSH GSA O O O O R' Ox R' R-SH-OH R-S-S-R R-S-N R-S-N R'' R'' O O O

Nagy P and Winterbourn CC., Adv. Mol. Toxicol, 4, 182-222, 2010. SchOxidationeme 1. Nom estatesnclature aofnd theOxid acysteinetion States osulfurf Comm on Derivatives of Cysteine

CySH CySOH CySO2H CySO3H ox state -2 0 +2 +4

sulfenic sulfinic sulfonic name thiol acid acid acid

O O CySSCy CySSCy CySSCy O ox state -1/-1 +1/-1 +3/-1 thiosulfinate thiosulfonate name disulfide ester ester Cysteine sulfenic acid

R-SH + H2O2 R-SOH + H2O

1. Very reactive short lived species.

2. We established estimated the rate constant for its reaction with thiols to give disulfides to be > 105 M-1s-1.

Cysteine sulfenic acid

R-SH + H2O2 R-SOH + H2O

1. Stable CySOH was characterized on: HSA, NADPH peroxidase, glutathione reductase, 1-Cys Prx and methionine sulfoxide reductase

2. Intermediate in 2-Cys Prxs, OhrR, cysteine-based proteases (Cathepsin B and L and SUMO proteases) and PTP-s

3. CySOH represent a major post-translational modification of periplasmic Cys residues of 1 Cys proteins. DsbG and DsbC maintain thiol homeostasis via reducing sulfenic acids. Depuydt, M. et al. Science 326 1109-1111, 2009 Cysteine disulfide

RSOH + R’SH RSSR’ + H2O

RSSR’ + R’’SH RSSR’’ + R’SH

1. Folding of newly synthesized polypeptides.

2. Spontaneous thiol exchange reactions are slow. Oxydoreductases catalyze the process in vivo. (PDI, Mia40, Dsb family) Mechanism for the folding process of polypeptides in the endoplasmic reticulum (ER) of eukaryotes.

H O O + H O 2 2 2 2 + 2O2 2O2 + 2H

Prx4 dimer 2 Prx4 monomers SH SH reduced PDI HS HS

S S E

SH E

r r

o o

1 1 O2 PDI HS S HS HS S SH FAD FAD

QSOX Disulfide SH S formation S SH SH S S

H2O2 PDI E

HS r o SH HSi 1 HS FAD Intramolecular thiol-disulfide interchange Cysteine disulfide

RSOH + R’SH RSSR’ + H2O

RSSR’ + R’’SH RSSR’’ + R’SH

1. Folding of newly synthesized polypeptides.

2. Spontaneous thiol exchange reactions are slow. Oxydoreductases catalyze the process in vivo. (PDI, Mia40, Dsb family)

3. Protein glutathionylation. Protection and signaling. (Grx, GsT, Gpx)

4. Disulfide formation on redox-active thiols is a dynamic and reversible process! (reduction by Trx, Grx, GR)

5. The redox status of the cell is controlled kinetically not thermodynamically! Higher oxidation states of Cysteine

R-SOH + oxidant RS-O2H + R-SO3H

1. Much less reactive.

2. Thought to be dead end oxidation products.

3. They are formed in competition with disulfide formation at higher oxidant concentrations Radical mediated oxidation of thiols (1 e- redox chemistry)

R-SH R' R'H

- R-S R-S R-S O2

R-S-S-R R-S-OO O2 R-SH

O2 R-S O R-SH R-S O R-S-S-R R-S-OOH R-S R-S R'-S H2O O OH R-S H2O2

O O O R-S-S-R' R-S-OH O2 H2O HO2 R-S R-S-OO R-S-OH

O O O

Nagy P and Winterbourn CC., Adv. Mol. Toxicol, 4, 182-222, 2010. Modeling cellular thiols as targets for H2O2

i

Winterbourn CC. Nat. Chem. Biol., 4, 278, 2008.

Prx 2

Prx 3 i Peroxiredoxins

Srx Redox switch

-S-S-

O S02H SH SOH

H2O2 H2O2

Pr-SH -S-S- Trx or Grx Reactivity of Prx3 Arg mutants Cys sulfur Reacting oxygen

~2 Å H ~1.5 Å H O ~1.6-2 Å O H

~1 Å Orders of magnitude Leaving oxygen Arg146 Arg123 drop in k

7 -1 -1 WT k ~ 2 x 10 M s eff

2 -1 -1 R146(G,K,H,A) keff ~ 2 x 10 M s 5

2 -1 -1 R123G keff ~ 7 x 10 M s 5

-1 -1 R123G/R146G keff ~ 2 M s 7

Nagy P et. al. JBC, 286, 18048, 2011. Facilitated oxidation of GAPDH active site

Cys by H2O2

k = 102 – 103 M-1s-1

Peralta D et. al. Nat Chem Biol, 2015 11, 156-63. A proton relay enhances H2O2 sensitivity of GAPDH to facilitate metabolic adaptation

140

(%) 120

100

80

60

40

20 Relative peroxide reduction rates by GAPDH by rates reduction peroxide Relative 0 WT C156S A proton relay enhances H2O2 sensitivity of GAPDH to facilitate metabolic adaptation

SH HS S S H2O2

SH HS SH HS

Trx (ox) Trx (red)

TrxR (red) TrxR (ox)

NAPH + H+ NADP+

G6PD Glucose-6-phosphate 6-phosphoglucono--lactone Evolutionary aspects of the proton relay system in GAPDH

k = 102 – 103 M-1s-1 The conserved Trp114 residue of TrxR1 has a redox sensor-like function triggering oligomerization and crosslinking upon oxidative stress related to cell death

Xu J et. al. Cell Death Dis, 2015 6, e1616. Phosphorylation pathways are redox regulated

SH SH PTEN + Oxidant PTEN SH SH Active form Inactive form Lee RS. et. al. Journal of Biological Chemistry, 273, 15366-72, 1998.

The activity of EGFR is also redox regulated!

797 797 EGFR- Cys + H2O2 EGFR- Cys-OH

A Cys797 equivalent is present in 9 other members of the EGFR family (including Her2, Her4)

Paulsen CE et. al. Nat Chem Biol, 2012 8, 57-64.

Modeling cellular thiols as targets for H2O2

1. Despite its very high abundance in the cytosol, reduced glutathione competes

very poorly for H2O2.

2. Owing to their large reactivity and local concentration, Prxs are favored

targets of H2O2.

3. Gpx1 and Prx consume virtually all

H2O2 until they are fully oxidized.

4. Based on the model, PTPs and other redox regulated enzymes, despite

their low pKa values, would only start reacting when Prxs are consumed.

Nagy P and Winterbourn CC., Adv. Mol. Toxicol, 4, 182-222, 2010. Peroxiredoxins

Srx Redox switch

-S-S-

O S02H SH SOH

H2O2 H2O2

Pr-SH -S-S- Trx vagy Grx Wood ZA et. al. Science, 2003 300, 650-3. Woo HA et. al. Cell, 2010 140, 517-28. Brown JD et. al. Cell Rep, 2013 5, 1425-35. Paulsen CE et. al. Nat Chem Biol, 2012 8, 57-64.

Proposed model for Prx-mediated redox signal transmission

Prx + H2O2 Prx-S-S-Prx

+ Kináz

Prx + oxidált Kináz Prx-S-S-Kináz

Nagy P. Antioxid Redox Signal, 2013 18, 1623-41. Sobotta MC et. al. Nat Chem Biol, 2015 11, 64-70. Acknowledgement

- Prof Kenneth Tew Prof. Elias Arner

- Prof. Kásler Miklós

- Éva Dóka, Pálinkás Zoltán, Bíró Adrienn, Budai Barna, Vasas Anita, Garai Dorottya, Ballagó Krisztina, Nagy Attila, Szűcs Judit, Lénárt Zsuzsanna MITO

- FP7 Marie Curie International Reintegration Grant

- OTKA kutatási pályázat

- Országos Onkológiai Intézet

Collaborators

Tobias Dick DKFZ, Germany

Elias Arner Karolinska Institute, Sweden

Christine Winterbourn Otago University, New Zealand

Christian Obinger BOKU University, Austria

John Wallace McMaster University, Canada

Martin Feelisch Southampton University, UK

Karol Ondrias Slovak Academy of Sciences i Michael Ashby Oklahoma University