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Protine PDF
Protine PDF
Dominique Mandon
INSTITUT DE CHIMIE
UMR CNRS 7177
Université de Strasbourg
4 rue Blaise Pascal
67070 Strasbourg cedex
FRANCE 1
Preliminary Note:
2
A multi-disciplinary area
3
A lively scientific area: main international meetings:
4
Useful references from which this course was setup
7
A matter of
curiosity:
No life without
Tansition Metals!
Only small amounts are required
Well-known
Essential functions:
8
9
10
Major economical outcomes
11
Industrial synthesis of Methanol
Lurgi and ICI process from natural gas (CH4).
Two steps
CH4 + H 2O CO + 3 H2
830°C, 18 bars = Reforming: syngas
CO + H2O CO2 + H2
Copper-based catalyst
CO2 + 3 H2 CH3OH + H 2O ∆rH°= - 68 kJ/mol
Reductase: Hydroxylase:
Electron transfer Where the reaction takes place !
Iron-containing enzymes:
13
Hydroxylase component in Iron-containing Methane MonoOxygenases:
14
Part 1: Principles of Bioinorganic Chemistry
Aliphatic lateral
chains
Proline contains a
Secondary amine function
16
Elemental components: amino acids - 2 -
17
Elemental components: amino acids - 3 -
18
Structure of proteins
19
Structure of proteins: secondary structure
20
Structure of proteins: secondary structure
α helixes β sheets
21
Structure of proteins: secondary structure: α helix
Internal stabilisation
α helixes
22
Structure of proteins: secondary structure: α helix
23
Structure of proteins: secondary structure: β sheet
24
Structure of proteins: secondary structure: β sheet
25
Structure of proteins: tertiary structure
1. Ionic interactions:
salt bridges
2. H- bonds
3. Van der Waals interactions
27
Part 1: Principles of Bioinorganic Chemistry
30 29
Magnetism and Transition Metals
31 30
Most frequently found geometry at active sites of metal-containing
biomolecules
32 31
Flexibility – Role of exogenous ligands
The properties differ from a given geometry to an other one
L
L
33
Various oxidation states !
34
Oxidation states and stability
35
Building-up a Frost Diagram
36
Fe Fe2+ + 2e- E° = -0.44 V [-0.88 V-eq]
37 36
Fe Fe2+ + 2e- E° = -0.44 V [-0.88 V-eq]
38 37
Fe Fe2+ + 2e- E° = -0.44 V [-0.88 V-eq]
39 38
Building-up a Frost Diagram
O2
H2O
40
O2 [FeO4]2-
H2/H+
Fe
Fe3+
Fe2+
41 40
Redox potentials can be tuned by geometry
If two oxidation states are presents, one of them being in a non-preferred geometry,
the redox potential can be dramatically shifted
His
Cu(I) Cu(II) + e- Cu
Met
His
Cys
44
Amino acids bind transition metals - 2 -
45
Direct coordination to metals – Active site 1
Protein
Protein
The heme can be bound to the protein by metal coordination via an amino acid residue (heme a, heme b),
alternatively by covalent bond (disulfide bridges in heme c). 47
Part 1: Principles of Bioinorganic Chemistry
Vibrational:
IR and Raman
EXAFS
UV-visible
Use of SPECTROSCOPIC TECHNIQUES
These techniques may be « time resolved »
(access to structural and reaction dynamics)
EPR
Mössbauer
Paramagnetic NMR 50
Vibrational:
IR and Raman
EXAFS
UV-visible
Use of SPECTROSCOPIC TECHNIQUES
These techniques may be « time resolved »
(access to structural and reaction dynamics)
EPR
Mössbauer
Paramagnetic NMR
51
UV-visible spectroscopy
52
Heme-containing porphyrins exhibit typical spectra: π -> π* transitions from the porphyrin macrocycle
Horse deoxyhemoglobin
53
Heme-containing porphyrins exhibit typical spectra:
π -> π* transitions from the porphyrin macrocycle
54
Iron / sulfur proteins are more difficult to detect by UV-visible
[4Fe-4S]2+
55
Vibrational:
IR and Raman
EXAFS
UV-visible
Use of SPECTROSCOPIC TECHNIQUES
These techniques may be « time resolved »
(access to structural and reaction dynamics)
EPR
Mössbauer
Paramagnetic NMR
56
A vibrational spectroscopic technique: Raman Resonance
Allows measurements in water – possibility to use polarized light – IR/RR => involvement of symmetry
Energy
Transmitted Radiation ν
hν h(ν0 − ν)
Incident Radiation ν0
Incident Radiation ν
= absorbed energy
?
IV
pH = 5.0
pH = 6.0
pH = 5.9
1/2
h k
∆E =
pH = 7.5
2π µ
pH = 8.4
mamb
µ=
ma+mb
UV-visible
Use of SPECTROSCOPIC TECHNIQUES
These techniques may be « time resolved »
(access to structural and reaction dynamics)
EPR
Mössbauer
Paramagnetic NMR
60
X-rays absorption technique: EXAFS
Extended X-ray Absorption Fine Structure
This technique is dedicated to structural studies in case no single crystals (for X-rays diffraction) can be
obtained: no crystallization, or study of unstable transient. Samples in frozen solution, and detection in
transmission or fluorescence.
I0
I0 I log
I
S
x
AF
EX
uil
se
uil
-se
M
pré
e-
hν
L
K λ
Irradiation yields so-called constructive and destructive oscillations, which patterns depend on
the oxidation state (pre-edge energy), as well as the environment of the sudied nucleus.
62
EXAFS
These oscillations yield an interferogram which can be simulated in order to get insight
into the structural parameters corresponding to the first coordination sphere.
Note: the oscillations parameters depend on the atomic number of the bound atom of the
ligand, and the technique is not accurate enough to unequivocally distinguish nearby lying
elements with similar distances, such as for instance, « N-metal» and « O-metal ».
Also, the angular data suffer from low accuracy.
63
IV
64
Vibrational:
IR and Raman
EXAFS
UV-visible
Use of SPECTROSCOPIC TECHNIQUES
These techniques may be « time resolved »
(access to structural and reaction dynamics)
EPR
Mössbauer
Paramagnetic NMR
65
EPR: Electron Paramagnetic Resonance
or Electron Spin Resonance (ESR)
66
Principle of EPR
Ĥ= gµBHŜz
∆E= gµBH
67
Electronic Zeeman effect:
degeneracy splitting of the α and β electronic spin states induced by a magnetic field
hν0
H=
gβ
hν0
g=
Hβ
For a given frequency ν, the field H is measured, yielding a g value different from that of the free electron,
being in that case characteristic of the studied system. 68
The spectrum is generally obtained as
the derivated form of the absorbtion data
For a given frequency ν, the field H is measured, yielding a g value different from that of the free electron,
being in that case characteristic of the studied system.
hν
g= 69
Hβ
First subtility: there may be an interaction between the electronic spin and a nuclear spin I:
for 1H, I = ½; for 14N, I = 1; for 63, 65Cu, I = 3/2.
The nuclear spin I can adopt 2I + 1 different orientations => 2I + 1 different contributions
. .
H3C radical: 4 lines, N-R-N : 5 lines, Cu(II): 4 lines A
70
Second subtility: anisotropy. Affects both g values and the hyperfine coupling
71
Second subtility: anisotropy. Affects both g values and the hyperfine coupling
x=z≠y axial
x≠z≠y rhombic
72
A suitable technique for copper-containing proteins
Plastocyanin
His
Cu
Met
His
Cys
CH
CH2
His
His Superoxide
N N His
dismutase
Cu
Zn
Asp His
His
73
Vibrational:
IR and Raman
EXAFS
UV-visible
Use of SPECTROSCOPIC TECHNIQUES
These techniques may be « time resolved »
(access to structural and reaction dynamics)
EPR
Mössbauer
Paramagnetic NMR
74
Paramagnetic NMR: nuclear spin / electronic spin interaction
75
Paramagnetic NMR
These differences are function of the « paramagnetism » of the molecule (HS / LS).
The magnetic susceptibililty in solution can be measured by 1H NMR (Evans method)
=> Acces to the spin state of a paramagnetic metal ion.
Once the magnetic properties of the metal centre are known, the measurement of
the chemical shifts and relaxation times allows insights into the structural
parameters of the active site, in particular the distance between the paramagnetic
centre and the nucleus which signal is measured.
76
Paramagnetic 1H NMR
Cytochrome c’, Fe(III) S = 5/2. Five-coordinate, His. Broad lines
III
77
Paramagnetic 1H NMR
Cytochrome c, Fe(III) S = ½. Six-coordinate, His + Met. Narrow lines
III
78
Vibrational:
IR and Raman
EXAFS
UV-visible
Use of SPECTROSCOPIC TECHNIQUES
These techniques may be « time resolved »
(access to structural and reaction dynamics)
EPR
Mössbauer
Paramagnetic NMR
79
Mössbauer Spectroscopy
Recoiless absorption of γ rays by 57Fe (also valid for 43 other elements of the periodic table)
Access to the oxidation sate of the metal, electronic environment and magnetic properties
When iron is complexed, this energy vary, and depends on the oxidation state
and coordination environment of the iron site.
=> Modulation of the γ rays energy. This is accomplished by Doppler effect
81
Historical setup
Current setup
δ, mm.s-1
82 79
δ: electronic density at the nucleus site.
δ depends on the nucleus radius changes between fundamental and excites states.
∆R
2 ∆R
δ∝ ∆ψ(0) ψ(0) <0
R
R
Zero coordinate: s electrons For Iron
1
EFG ∝
r3 83
Isomer shift Quadrupole splitting Hyperfine Magnetic
δ ∆Eq Interaction
Energy released by Exc. => Fund. Electric Field Gradiant Internal or external magnetic
Oxidation State Metal- ligand interaction interaction
Ie = 3/2
Ie = 3/2
Ig = 1/2 Ig = 1/2
δ δ, ∆Eq 84
H applied
(ou B)
Tentative prediction of the oxidation and spin states of iron-containing centres
1.0
Fe(II)
S=2
0.5 Fe(II)
Fe(III) S=0
δ [mms-1]
S = 5/2
Fe(III)
S = 1/2
0 1 2 3
∆EQ [mms-1] 85
Oxidation state of iron: Fe – oxygen, Fe=O
IV
Vibrational:
IR and Raman
EXAFS
UV-visible
Use of SPECTROSCOPIC TECHNIQUES
These techniques may be « time resolved »
(access to structural and reaction dynamics)
EPR
Mössbauer
Paramagnetic NMR 87
88