Thiol Proteins

Thioredoxin, glutaredoxin, protein disulfide isomerase,peroxidases, methionine sulfoxide reductase

Thiol Protein – at least one very reactive cysteine

Thiols in Biology

COO - COO -

H-C-CH2-SH H-C-CH2-S- + H+ pKa = 8,37 NH3

+ NH3+

pH = 8,37: [RSH] = [RS-] thiol thiolate

pH < 8,37 [RSH] > [RS-]pH > 8,37 [RSH] < [RS-]

cysteine

Glutathione (GSH)

glycinecysteinel-glutamate

CH2 CH2 C

O

CH

NH3

CO

OCCNH

O

CH2

H

SH

NH CH2 CO

O

pKa = 9,2

Low toxicity

High intracellular concentrations (1-10mM)

Thiolate stabilization in proteins

R1SH + R2SS R2 R1SS R2 + R2SH

Thiol/disulfide exchange reactions

central theme in biology

-structure of proteins

-Regulation of protein activity (enzyme, transcription factors...)

-cellular redox homeostasis etc...

Rlg Rn

Rn-S- + S-S S-S + Rlg-S- R Rnucleophilicagent leaving group

k

V = k[Rn-S- ] [ RSSRlg]

Mechanism of thiol/disulfide exchange reactions

nucleophilic substitution

Thioredoxin (Trx)Dissulfide reductase - 12-13kDa - plants, animals, yeast , bacteria

thioredoxin reductase

NADPH + trx (-SS-) NADP + trx (-2SH)

glutathione reductase

NADPH + GSSG NADP + 2 GSH

Cys35-SH

Cys32-S -

trx

S

S

Cys35-S

Cys32-S -

H

-S

S

HS

HS

Cys35-S

Cys32-S

targettrx target

Cys35-SH

Cys32-S -

trx

S

S

H

-S

S

HS

HS

Cys35-S

Cys32-S

trx target

Thioredoxin fold

thioredoxin

Grx GSSG

GSSG reductase NADPH,H+

2 GSH

)

(2-RSH)

(RSSR)

Grx

RibonucleotideReductase,other targetoxidized

RibonucleotideReductase,other targetreduced

Glutaredoxin (Grx)

12-13kDa CXXC motifi (CPYC)

Trx and Grx targetsyeast

Oxidized Grx1 GSSG Trx1Target Grx2 Trx2

(2-RSH) (2-RSH)

Glr1, NADPH, H + Trr1, NADPH, H +

Reduced Grx1 Trx1Target Grx2 GSH Trx2

(-SS-) (-SS-)

RibonucleotideReductase

PAPSreductase

Tioredoxinperoxidase

Methioninesulphoxidereductase

???

Methionine sulfoxide reductase

Reviewed by Weissbach et al. (2002) Arch. Biochem. Biophys., 397:172.

Deglutathionylation by Grxmonothiol mechanism

Grx-S- + protein –SS G Grx – SS G + protein-SH

Grx – SS G + GSH GSSG + Grx-S-

Thiol/disulfide oxido-reductases

Protein Motif in active site Redox Potential (mV)

Trx Cys-Gly-Pro-Cys -270

Grx Cys-Pro-Tyr-Cys -200 to –235

Tryparedoxin Cys-Por-Pro-Cys -249

Protein disulfide isomerase Cys-Gly-His-Cys -127(PDI)

DsbA Cys-Pro-His-Cys -125

PDI and DsbA generate disulfide bonds in proteins

PDI is in the endoplasmatic reticulum (ER)DsbA in periplasm (bacteria)

GSH:GSSG in ER 1:1 to 3:1(100:1 to 30:1 in cytoplasm)

Thiol proteins oxidation states

Protein Oxidation States Trx Cys-Gly-Pro-Cys -2 (thiol), -1 (disulfide)

Grx Cys-Pro-Tyr-Cys -2 (thiol), -1 (disulfide)

Tryparedoxin Cys-Por-Pro-Cys -2 (thiol), -1 (disulfide)

Protein disulfide isomerase Cys-Gly-His-Cys -2 (thiol), -1 (disulfide)(PDI)

DsbA Cys-Pro-His-Cys -2 (thiol), -1 (disulfide)

Glutathione Peroxidase Asn-Val-Ala-Ser-Lys-Cys -Gli -2 (thiol), 0 (sulfenic acid)non-selenium (GPx)

Prx = peroxiredoxin

2 RSH + H2O2 RSSR + 2 H2O2 RSH + ROOH RSSR + ROH + H2O

197 residues - 25kDa

Active site: cysteine 47cysteine 170 – catalysis

Netto et al. (1996) J. Biol. Chem., 271, 15315-15321 .

Type A YPxDF[T/S]FVCPP[T/S]E[I/L/V] .....C-terminal VCrP

Type B HPxDFTPVCPTTE

Type C YPx[A/D]xTP[G/V] CPTx[Q/E]xCrx[F/L]

Type D xP[G/A]A[F/Y][T/S][P/G]xCP[S/T]xxHxP

Type E xP[D/S]DTxVCPxx[Q/S]x[K/R]

Trivelli, X., Krimm, I., Ebel, C., Verdoucq, L., Prouzet-Mauléon, V., Chartier, Y., Tsan, P., Lauquin, G., Meyer, Y., Lancelin, J. (2003) Characterization of the yeast peroxiredoxin Ahp1 in its reduced and overoxidized inactive forms using NMR. Biochemistry 42: 14139-49.

TrxPrx

Prx

Gpx

Trx = Prx = Gpx

Amino acid sequence

Choi et al. (1998) Nature Struct. Biol. 5: 400

Prx and Trx have the same fold

Peroxynitrite reductase activity of bacterial Prx

Bryk, R., Griffin, P. & Nathan, C.Nature (2000), 407:211-215

OONO - + H+ + Trx NO2 + H2O + Trx 106 M-1 s-1 SH S- S -- S

Prx

Sp-

SH

47

170

ROOH ROH

SOH

SH

HS

-S

HS

-Sp

RSSR

170

47

S

SH

S

-S

H2O

2 RSH

Sp-

SH

47

170

ROOH ROH

SOH

SH

2 RSHRSSR

A. B.

C.

S

S

RSSR

Sp-

SH

62

120

ROOH ROH

SOH

SH

H2O2 RSH

2-cys típica

2-cys atípica

1-cys

61

125

SH

SH

61

125

SOH

SH

ROOH

Reduced protein

61

125

S

S+ H2O

disulfide

fast

61

125

SO2H

SH

ROOH

Sulfinic acid(oxidation state = +2)

ROOH61

125

SO3H

SH

Sulfonic acid(oxidation state = +4)

+ DTT

61

125

SS-DTT-SH

SH

+ DTT (-SS-)

Sulfenic acid

61

125

SSG

SH

GSHGSH

GSSG

Glutathionylatedprotein

Ritz et al (2001) Science 294: 158

Prx (AhpC) as GSSG reductaseMutation = one amino acid insertion (Phe)

Versatility of Prx

Crystal structure of oxydized decamer High concentrations

Disulfides in yellow

Thioredoxin fold:2x (beta-alfa-beta)

Heat shock (43 C por 30’)

plating

WT

Delta prx1/prx2

Delta prx1/prx2 + pRS416/cTPxI

Delta prx1/prx2 + pRS416/C47S/C170S

2-Cys –Prx are also chaperons!!

Proteins analyzed by SECcTPxI,cTPxII

40-1000 kDa!!MW = 21,5 kDa(2-50 proteínas)

Western blot 10%PAGE

Particle diameter: 22-28nm

FI fraction

Electron Microscopy (EM)

Two views:

409 “End on”(five fold symmetry)

170 “Double dot”

FII fraction

Electron Microscopy (EM)

Particle diameter: 14 nm

P-S47H R-S47- P-S47OH

INATIVA REATIVA INSTÁVEL

R-SHP-S47 - S170-P

peróxido

P-S47O2H

R-SH

oligomerização

oligomerização

P-S47H R-S47- P-S47OH

INATIVA REATIVA INSTÁVEL

R-SHP-S47 - S170-P

peróxido

P-S47O2H

R-SH

srx

Sulfiredoxin (Srx) – 13kDa

Cys 84 – reactive site

Homolog in humans

Biteau et al. (2003), Nature 425: 980

Number of genes >>>>> Number of folds

TrxGrxGSH transferaseGSH peroxidasePrxCalsequestrins

PDI Methionine sulfoxide reductase

Trx fold

Trx X Cytochrome c Maturation Protein (CMP)

- high amino acid sequence divergence

- trx fold

- Conserved residues involved in catalysis

Motif analysisTrx, CMPs and Prx common ancestor !!

New peroxiredoxin from Xylella fastidiosa ?

Ohr = Organic Hydroperoxide Resistance protein

Deletion of Ohr gene:

- Bacteria sensitive to organic peroxides (not H2O2!!)

- Only organic peroxides induce transcription of Ohr gene

pathogen ROSRNS

PLANT

A. B.

0

5

10

15

20

OHR C125S C61S

Initi

al r

ate

(uM

/min

)

0

5

10

15

20

25

30

OHR C125S C61S

Initi

al r

ate

(uM

/min

)

Cussiol J.R.R., Alves S.V., Oliveira M.A. e Netto L.E.S. (2003) J. Biol. Chem , 278, 11570—11578

TBHP (200 M) H2O2 (200 M)Ohr (2 ng/ul) Ohr (50 ng/ul)

Ohr is a thiol — dependent peroxidase

Ohr - Xylella fastidiosa

Tratada com t-BOOH (1mM/RT/1h)Tampão – Tris-Cl pH=8,5 0,1MPrecipitante – PEG 4000 25%

Complete dataset at 1.9 Å

Oliveira et al., (2004) Acta Crystall. D60, 337

No trx fold – alfa-beta fold

Ohr Prx GSH px. New class of thiol dependent peroxidase

Ohr structure