thiol proteins thioredoxin, glutaredoxin, protein disulfide isomerase,
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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
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