Marc Fontecave

Laboratoire de Chimie des Processus Biologiques, UMR 8229 CDF/CNRS/UPMCCollège de France, 11 Place Marcelin Berthelot, 75231 Paris Cedex 05

mfontecave@college-de-france.fr; Phone: (0033)144271360

Fer et Soufre: Un mélange bio-inorganique très réactif

Iron-Sulfur Clusters

Exotic Iron-sulfur assemblies

Fe SS

Fe

S

FeS

FeS

S S

SCys

Cys

Cys

Cys

NiS

S Ni NN

Cys

Cys

SCys

FeSS

Fe

S

NiS

FeS

S S

SCysCys

Cys

Cys

S

Fe

His

NN

CO dehydrogenase

Acetyl-CoA Synthase

J.W. Peters, J.B. Broderick et al. Biochemistry 2014 53 4090

Hydrogenase

Exotic Iron-sulfur assemblies

Exotic Iron-sulfur assemblies

Nitrogenase

greigite

Fe(Ni)S centers of proteins: inventions of the biological world or relics of mineral ancestors capable of catalytic activity ?

pyritemackinawite

Fe SS

Fe

S

FeS

FeS

S S

SCys

Cys

Cys

Cys

NiS

S Ni NN

Cys

Cys

Correlation motif CX 2CX2CX3C (120 genomes):-Methanogenesis-Archaea-Anaerobiosis-hyperthermophily

From archaea to human beings

0 -200 -400 -600+200+400+600+800

Cu2+/1+O2/H20

H+/H2

Cytochromes Fe3+/2+

rubredoxin

ClustersFe-S

[3Fe-4S]

[2Fe-2S]

[4Fe-4S]

Redox potentials of biological metal centers

Iron-Sulfuromics

�How many FeS proteins in an E. coli cell ?�Relative abundance of the various clusters ?�How many different binding motifs ?

~ 110 [Fe-S] proteins (~ 70 systems)(potentially 200)

-Electron transfer: 55%-Non Redox: 17%-Redox (Radical-SAM): 10%-Sensor/Regulator: 6%-Structural: < 4%-Cluster biosynthesis 8%

[2Fe-2S]: < 10% [4Fe-4S]: > 90%

More than 40 signatures !CX2CX2CX3C (72), CX2CX4CX3C (20), CX2CX2CX7C (16),…….

M. Fontecave, Nature Chem. Biol. 2006

Respiration

TCA cycle

Gene regulationRNA modification

DNA repair

FnrYeiL IscRNsrR SoxRRsxBRsxC AcnA

AcnBFumAFumBSdhB

[2Fe-2S] [4Fe-4S]

MutYNth

DmsADmsBFdnGFdnHFdhEFdhFFdoGFdoHFrdB GlpBGlpC

HyaA HybAHybOHycBHycFHycGHydNHyfAHyfHHyfI

NapANapFNapGNapHNarGNarHNarYNarZNirBNuoBNuoENuoFNuoGNuoINrfC

MiaBRumARumBRlmN

SR

RS

SR

RSRS SR

SRRS

FadH (FA)IscA (FeS)IscU (FeS)Fdx (FeS)SufA (FeS)SufB (FeS)ErpA (FeS)NfuA (FeS)

Biosynthetic pathwaysBioB (Biotin) ThiH (Thiamin)NadA (NAD) LipA (Lipoate)GltB (Aa)GltD (Aa)IlvD (Aa)LeuC (Aa)HemN (Heme)IspG (IPP DPP)IspH (IPP DPP)MoaA (Mo-co)

Fe, S, N metabolism

FhuFBfDCysIHcpHcr

Post-translational modification

AslBNrdGPflAYijMYbiYRimO

Other cellular processes

GlcFHcaCHcaESdaASdaBTdcG

FixXPaaE TtdATtdBXdhCXdhD

Unknown

AegAYagTybhJYcbXYccMYdeMYdePYdhVYdhXYdhYYdiJYdiTYdbKYeaWYeaXYeiAYeiTYfaEYfhLYgcF

YgcOYgfKYgfSYgfTYggWYgiQYhcCYjeKYjeSYjjWYkgFYnfEYnfFYnfGYsaA

The computational model was then applied to over550 prokaryotic genomesto screen foriron–sulfur proteomes; the results are publicly available at:http://biodev.extra.cea.fr/isph.This study represents a proof-of-concept for the application of a penalized linear model toidentify metalloprotein superfamilies on a large-scale. The application employed here, screeningfor iron–sulfur proteomes, provides new candidates for further biochemical and structuralanalysis as well as new resources for an extensiveexploration of iron-sulfuromes in themicrobial world

Metallomics 2014, 6, 1913–1930

�Transport et transfert d’électrons (photosynthèse, respiration,…) (1960….)

e- + [xFe-xS]2+ [xFe-xS]+

Complexe I from Thermus thermophilus7 subunits, 9 FeS clusters (Science 2006)

Hydrogénase H2 2H+ + 2e-

Iron-sulfur clusters in Biology

Aconitase

HOOC-CH2-C-CH2COOH

OH

COOH

HOOC-CH=C

CH2COOH

COOH

- H2O

Citrate

aconitate

B H

�Catalyse non rédox (dehydratases, ACONITASE ,…) (1970….)

Iron-sulfur clusters in Biology

�Modulation of gene expression (FNR, SoxR, IRP,…) (1980….)

IRP « apo »: se fixe sur l’ARNmIRP « Fe-S »: ne se fixe pas sur l’ARNm

IRP: Iron Regulatory protein

Iron-sulfur clusters in Biology

�Transport et transfert d’électrons (photosynthèse, respiration,…) (1960….)

e- + [xFe-xS]2+ [xFe-xS]+

�Catalyse non rédox(déhydratases, ACONITASE ,…) (1970….)

� Modulation de l’expression des gènes(FNR, SoxR, IRP,…) (1990….)

�Catalyse rédox(enzymes Radical-SAM) (2000…)

SAM:S-adenosylmethionine

Iron-sulfur clusters : ever expanding rolesM. Fontecave

Nature Chemical Biology 2006, 2, 171-174

Iron-sulfur clusters in Biology

Radical-SAM Enzyme Superfamily

Biosynthesisof:-Cofactors (lipoate, PQQ, molybdopterin…)-Antibiotics (desosamine, mitomycine, fosfomycine,…)-Vitamins (biotin, thiamin,…)-Alkaloïdes-ChlorophylleMetabolism of:-Sugars-Amino-acids-HydrocarbonsModification of-RNA (tRNA, rRNA,..)-Enzymes (hydrogenase, nitrogenase, RNR,PFL…)Repair of:- DNA

SAMCX3CX2C

HydE

Y. Nicolet, M. Fontecave, J. C. Fontecilla-Camps et alJ. Biol. Chem. 2008, 283, 18861-18872

Adenosylmethionine as a source of 5’-deoxyadenosyl radicalsM. Fontecave, E. Mulliez, S. Ollagnier-de ChoudensCurrent Opinion in Chemical Biology 2001, 5, 506-511

S-Adenosylmethionine-dependent radical-based modification of biological macromoleculesM. Atta, E. Mulliez, S. Arragain, F. Forouhar, J. F. Hunt, M. FontecaveCurr. Op. Struct. Biol. 2010, 20, 684-692

Radical SAM, a novel protein superfamily…H.J. Sofia et alNucleic Acid Res. 2001, 1097-1106

RH: substrate

RH

R.

The « Radical-SAM» family:

A radical chemistry

Nature Chemical Biology 2013, 9, 333-338

RimO-S12 complex:a model

CH2

C

CH

O-O

CH

C

CH

O-O

SH3C

ribosomal protein S12 [D88]

RimO (yliG)

SAM, "S"

CH3S Fe

+

2+RH

SAM

SFe

2+

2+RH

Ado°

CH3S Fe

S

2+

2+R°

AdoH

CH3S Fe

S

2+

2+RSCH3

e-

AdoH

R-HNCH C

C

R

O

CO2H

H

H

Ado° AdoH

R-HNCH C

C°

R

O

CO2HH

CH3S°

R-HNCH C

C

R

O

CO2H

H3CS

H

RimO

Iron-sulfur centers: biosynthesis and repair

FeOn(OH)p

Fe ?

« S »

Cys-SH

[Fe-S]Fe-S

Fe-S Fe-S

stress

repair

regulation

E. Coli cell

ISCSUF

maturation

SufE

SufD

SufB

SufS

L-cysteine L-alanine

S-

Cys

S-Cys

S-ATP

ADP + Pi

S

sufA sufB sufC sufD sufS sufE

G. Layer, S. Aparna Gaddam, S. Ollagnier-de-Choudens, D. Lascoux, M. Fontecave, F. Wayne OuttenJ. Biol. Chem. 2007, 282, 13342-13350

L. Loiseau, S. Ollagnier-de-Choudens, L. Nachin, M. Fontecave, F. Barras J. Biol. Chem. 2003, 278, 38352

S. Ollagnier-de-Choudens, M. Fontecave et al Febs Letters 2003, 555, 263

S. Ollagnier-de-Choudens, M. Fontecave, F. Barras et al J. Biol. Chem. (2003) 278, 17993

Cysteinedésulfurase

ATPase

L. Nachin, F. Barras et al. , EMBO J. (2003) 22 427

S. Ollagnier-de-Choudens, M. Fontecave et al J. Am. Chem. Soc. 2009, 131, 6149

SufC

SCAFFOLD

FADH2

N

N

N

N

O

O

H3C

H3C

H

R

H

H Flred

Marc Fontecave

Laboratoire de Chimie des Processus Biologiques, UMR 8229 CDF/CNRS/UPMCCollège de France, 11 Place Marcelin Berthelot, 75231 Paris Cedex 05

mfontecave@college-de-france.fr; Phone: (0033)144271360

Fer et Soufre: Un mélange bio-inorganique très réactif

+ L-Cysteine+ CyaY-Fe3+

SufESufS SufESufS

SufC

SufB

SufD

SufC

4Fe-4S

Apo-aconitaseHolo-aconitase

SufC

SufB SufD

SufCFADH2

Fe-SSufA

SufA

Holo-proteinApo-protein

Flavin reductase

NAD(P)H

NAD(P)+

FAD

L-alanine, CyaY

FAD

SufC

SufB SufD

SufCFADH2

SufC

SufB SufD

SufC

1 2

3

4

5

Iron-sulfur Cluster Assembly: the SufBCD complex is a new type of Fe-S scaffold with a flavin redox cofactorS. Wollers1, G. Layer, R. Garcia-Serres, L. Signor, J.-M. Latour, M. Fontecave, S. Ollagnier de ChoudensJ. Biol. Chem. 2010, 285, 23331-41

Electron transfer (52)Ferredoxins (fdx, bfd, yfaE)Succinate dehydrogenase (sdhB)Sulfite reductase (cysI)Nitrite reductase (nirB)Formate-dependent nitrite reductase (nrfC)2,4-dienoyl-CoA reductase (fadH)DMSO reductase (dmsA, dmsB)SoxR reductase (rseC, rsxB, rsxC)Formate dehydrogenase N (fdnG, fdnH, fdhD)Formate dehydrogenase H (fdhF)Formate dehydrogenase O (fdoG, fdoH)Ferrichrome reductase (fhuF)Fumarate reductase (frdB)Glutamate synthase (gltB, gltD)Hybrid cluster protein (hcp)HCP reductase (hcr)Periplasmic nitrate reductase (napA, napF, napG, napH)Respiratory nitrate reductase 1 (narG, narH)Respiratory nitrate reductase 2 (narY, narZ)NADH:quinone oxidoreductase (nuoB, nuoE, nuoF, nuoG, nuoI)Anaerobic glycerol phosphate dehydrogenase (glpB)Hydrogenase 1 (hyaA)Hydrogenase 2 (hybO, hybA)Hydrogenase 3 (hycB, hycF, hycG)Hydrogenase 4 (hyfA, hyfH, hyfI, hyfR)Xanthine dehydrogenase (xdhC)

Metallosite assembly (7)Scaffold proteins (iscU, nfuA)A-type transporters (iscA, sufA, erpA)Mo-co biosynthesis protein (moaA)SufB (sufB)

Nucleic acid-binding proteins (7)- gene expression regulators (4)Fumarate and nitrate reduction regulator (fnr)Isc regulator (iscR)Redox sensitive transcriptional activator (soxR)Transcriptional activator (yeiL)- DNA repair (2)Adenine glycosylase (mutY)Endonuclease III (nth)- RNA modification (1)RNA 5-methyluridine methyl transferase (rumA)Redox catalysis (12)- Radical-SAM (10)Biotin synthase (bioB)Coproporphyrinogen synthase (hemN)Lipoate synthase (lipA)Thiazole biosynthesis protein (thiH)RNA thiomethyl transferase (miaB)Anaerobic ribonucleotide reductase activating component (nrdG)Pyruvate-formate lyase activating component (pflA)Sulfatase maturase (aslB or atsB)Methylthiotransferase (rimO)- Others (2)4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (ispG)4-hydroxy-3-methylbut-2-enyl diphosphate reductase (ispH)

Non redox catalysis (10)Aconitases (acnA, acnB)Fumarases (fumA, fumB)Dihydroxyacid dehydratase (ilvD)Isopropylmalate dehydratase (leuC)Quinolinate synthase (nadA)Serine-threonine deaminase (sdaA, sdaB)Serine dehydratase (tdcG)