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Struct. and funct. study of mR machinery involved in biosynth and insertion of the organoMe+ active site of [FeFe]-H2ase

the subject of my thesis and some of my "achievements"
by

Roman Rohac

on 9 March 2016

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Transcript of Struct. and funct. study of mR machinery involved in biosynth and insertion of the organoMe+ active site of [FeFe]-H2ase

second year PhD student of the IRTELIS graduate program of the French CEA
Institut de Biologie Structurale - Grenoble

Structural and functional study of molecular machinery
involved in biosynthesis and insertion of the organometallic
active site of [FeFe]-hydrogenases

by roman rohac
GTPase activity scaffold protein - insertion of H-cluster
3QQ5, Cendron, L. et al, 2011
WHAT?
1HFE, Fe-only hydrogenase
from Desulfovibrio desulfuricans
Nicolet, Y. et al, 1999
FeFe-hydrogenase maturase
HydE
from Thermotoga maritima
3CIX, Nicolet, Y. et al., 2008
FeFe-hydrogenas maturase
HydG
uses Tyrosine to produce CO and CN- ligands
[NiFe]-hydrogenases, [Fe]-hydrogenases and
[FeFe]-hydrogenases

in FeFe-hydrogenases catalytic H-cluster = cubane Fe4S4 cluster
linked to a modified Fe2S2 subcluster with non-proteic ligands
hydrogenases = reversible oxidation of molecular hydrogen
unknown structure
unknown substrate
active form structure unknown
insertion mechanism unknown
radical SAM enzymes
catalytic H-cluster from FeFe-hydrogenase
CO & CN- ligands
dithiomethylamine
DTN
CO & CN-
FeFe-hydrogenase maturation
WHY?
HOW?
X-ray crystallography and biochemistry under reducing conditions
iron-sulfur clusters very sensitive to oxygen
all experiments performed under controlled atmosphere = 95% Nitrogen & 5% Hydrogen
cell culture - harvesting - protein purification - biochemical assays - crystallization - flash freezing - data collection
ö
ligand binding assays and investigation of paramagnetic cluster environement:
liquid state NMR of proteins with paramagnetic FeS clusters

FeS characterization by spectroscopic methods:
M ssbauer, Raman, UV-visible, Electron Paramagnetic Resonance
Collaboration
Hydrogen - clean energy of the future ?
Excitement..
..Beauty
plausibly ones of the very first enzymes
CO
HCN
CO
H
2
S
2
CH COSH
3
H O
2
Results
Ph.D. Supervisor: Yvain Nicolet
Ph.D. Director: Juan Carlos Fontecilla-Camps

What's next ?
Tyrosine
unknown
2
CH COSCH
3
2 H O
2
O
2
4 H
+
e
-
e
-
H
+
H
+
PS II
PS I
e
-
b6f
e
-
e
-
2 H
+
H
2
H
+
H
+
F1
ADP
P
i
ATP
H S
2
N
2
O -tolerant hydrogenase
2
Full transcript