pregunta 2: por qué sirve la epr espectroscopía tan bien...
TRANSCRIPT
Pregunta 1: Qué es esto?
1
Pregunta 2: Por qué sirve la EPR Espectroscopía tan
bien para el empleo en Biología, Bioquímica y Medicina ?
Facultad de Quimica, UNAM
Resonancia Paramagnética Electrónica (RPE/EPR)
Aplicación en Biología, Bioquímica y Medicina (= BIOEPR) 27.10. y 3.11. 2016
Peter M.H. Kroneck
Helmut Beinert 1913 - 2007
2
Richard Sands
Conferencias 27.10. y 3.11. 2016
(Scope of the Lectures)
• Por favor no dude en hacer preguntas!
• Algunas repeticiones
• Metales en proteínas - „Complejos inorgánicos
simples“ y objetos de investigación ideales para
EPR (Continuous Wave EPR = CWEPR)
• Historia - Metales en proteínas y EPR, una
estrecha relación: Keilin, Malmström, Beinert
• EPR de Cu, Fe & Mo Proteínas seleccionado
• Perspectiva -Técnicas avanzadas
3
Introducción a/Introduction to BIOEPR
R. R. Crichton, 2012 Biological Inorganic Chemistry – An Introduction, Elsevier, Amsterdam
H.M. Swartz, J.R. Bolton, D.C. Borg, 1972 Biological Applications of Electron Spin Resonance, Wiley Interscience, New York
J.R. Pilbrow, 1991 Transition Ion Electron Paramagnetic Resonance, Oxford University Press, USA
Dalton Transactions, 2006
4415-4435, W. R. Hagen EPR spectroscopy as a probe of metal centres in biological systems
W.R. Hagen, 2009 Biomolecular EPR Spectroscopy, CRC Press, Boca Raton, Florida
www.bt.tudelft.nl/biomolecularEPRspectroscopy
Inorganic Electronic Structure and Spectroscopy, 1999 (advanced book)
(eds E.I. Solomon, A.B.P. Lever), John Wiley & Sons, LTD
4
EPR of Metalloproteins - Classics
Proc. R. Soc. Lond. A, 1957 (Heme-Iron)
Vol. 240, 67-82, J. E. Bennett, J. F. Gibson, D. J. E. Ingram Electron-Resonance Studies of Haemoglobin Derivatives. I. Haem-Plane Orientation
Nature, 1959 (Copper)
Vol. 183, 321-322, B. G. MALMSTRÖM, R. MOSBACH, T. VÄNNGÅRD
An Electron Spin Resonance Study of the State of Copper in Fungal Laccase
Biochem. Biophys. Res. Commun, 1961 (Iron-Sulfur)
Vol. 5, 40-45, H. Beinert, W. Lee Evidence for a New Type of Iron Containing Electron Carrier in Mitochondria
Proceedings National Academy of Sciences (USA), 1964 ([2Fe-2S] Ferredoxin)
Vol. 52, 1263-1271, Y. I. SHETHNA, P. W. WILSON, R. E. HANSEN, H. BEINERT IDENTIFICATION BY ISOTOPIC SUBSTITUTION OF THE EPR SIGNAL AT g = 1.94 IN A NON-HEME IRON PROTEIN FROM
AZOTOBACTER
Nature, 1966 (Molybdenum)
Vol. 212, 467-469, R.C. Bray, L.S. Meriwether Electron Spin Resonance of Xanthine Oxidase Substituted With Molybdenum-95
Proceedings National Academy of Sciences (USA), 1981 (Manganese)
Vol. 78, 274-278, G. C. DISMUKES, Y. SIDERER Intermediates of a polynuclear manganese center involved in photosynthetic oxidation of water 5
EPR websites - 1
6
Daniella Goldfarb, Weizmann Instiute of Science, Israel
https://www.weizmann.ac.il/chemphys/EPR_group/
Gunnar Jeschke, ETH Zürich, Switzerland
http://www.epr.ethz.ch/
Robert Bittl, FU Berlin, Germany
http://www.physik.fu-berlin.de/einrichtungen/ag/ag-bittl/mitarbeiter/bittl/index.html
Wolfgang Lubitz, Frank Neese, MPI CEC, Mülheim, Germany
https://cec.mpg.de/1/home/ (ORCA Software; ORCA Workshops)
Wilfred Hagen, TU Delft, The Netherlands
http://www.tnw.tudelft.nl/en/about-faculty/departments/biotechnology/people/
biocatalysis/profdr-wr-hagen/
Brian Hoffman, Northwestern University, USA
http://www.chemistry.northwestern.edu/people/core-faculty/profiles/brian-
hoffman.html
EPR websites - 2
7
National Biomedical EPR Center, Medical College of Wisconsin, USA
http://http://www.mcw.edu/EPR-Center.htm
EPR Center, University of Denver (The Eatons), USA
http://epr-center.du.edu/
ESR Group, Royal Society of Chemistry, UK
http://www.esr-group.org/
Bruker
https://www.bruker.com/
Center for EPR Imaging, The University of Chicago, USA
https://epri.uchicago.edu/page/epr-imaging
International EPR (ESR) Society (IES); EPR newsletter
https://www.ieprs.org/; http://www.epr-newsletter.ethz.ch/
Espectroscopía/Spectroscopy-Energía/Energy
4 - 1 eV 8000 2000 0.1-0.01 10-4 -10-5 10-6 -10-7
X-ray UV/vis Infrared Microwave Radio
30000 25000 20000 15000 10000
Wavenumber (cm-1)
2500 3000 3500
Magnetic field (G)
1800 1900 2000 21001400 1500 1600 1700
Wavenumber (cm-1)
Q
0 mm/s
EQ8960 8980 9000 9020 9040 9060
Energy (eV)
pre-edge
edge
near-edge
EXAFS
11 12 13 14 15 16 17
Frequency (MHz)
400 500 700 800
351
676
568
530
476
407
Raman Shift (cm-1)
x1/3
Gamma
EPR ENDOR
NMR
IR
Raman
ABS
MCD
CD
XAS
EXAFS
Möss-
bauer
14000
8
EPR y NMR son métodos diferentes
electron proton ratio
Rest mass me =9.1094*10-28 g mp =1.6726*10-24 g 5.446*10-4
Charge e=-4.80286*10-10ESU e=4.80286*10-10ESU -1
Angular momentum h/4p h/4p 1
Magnetic dipole
moment
mS=-ge meS
ge= 2.002322
me=eh/4pmec =
9.274*10-21 erg/G
mS=-gN mNS
gN= 5.5856
mN=eh/4pmNc =
5.0504*10-24 erg/G
1836.12
Frequency: Factor 1000 larger in EPR ! (GHz instead of MHz)
Coupling strength: Factor 1 000 000 larger in EPR ! (MHz instead of Hz)
Relaxation Times: Factor 1 000 000 smaller in EPR ! (ns instead of ms)
= much higher techniqual requirements, but unique sensitivity to molecular motion
Sensitivity : Factor 1 000 000 better than in NMR !! (1nM vs 1mM )
An ideal case, though 9
Alfred Werner (University of Zürich, Nobel Prize
in Inorganic Chemistry, 1913)
Co[(NH3)6]Cl3 Co[(NH3)5Cl]Cl2
Recordar: Color y Magnetismo
10
Recordar: Color y Magnetismo Estados de la vuelta variables de centros metálicos
OR
High-Spin, S= 5/2 Low-Spin, S= 1/2
Depending on the METAL ION ENVIRONMENT, balance of Crystal
Field Splitting, 10Dq and Spin-Pairing Energy, P
10Dq <
Weak Field
High Spin
10Dq >
Strong Field
Low-Spin
For a d5 configuration, Fe(III)
11
Recordar: Molecular Orbital Theory - Experimental proof of covalency - EPR
2500 3000 3500
g||= 2.262
g|_= 2.047
A||(Cu)= 540 MHz A
||(N)= 40.6 MHz
A|_(Cu)= 81 MHz A
|_(N)= 38.2 MHz
sim
exp
Magnetic field (G)
[Cu(imidazole)4]2+
Crystal Field Picture
Observed Hyperfine Coupling between magnetic moment of
the Cu electron spin and the magnetic moment of the nuclear spin
of 4 nitrogens (I=1)
Pure electrostatic interaction between the
ligands and metal, the ligands being
regarded as negative point charges.
No coupling expected
N N
N N
12
25000 20000 15000 100000
2
4
(
mM
-1 c
m-1
)
Magnetic field (G)
Wavenumber (cm-1)
EPR
ABS
2800 3000 3200 3400
Química Biológica Inorgánica – Objetivo
Estructura (3D/Electrónica)→ Función → Mecanismo
13
R Medicine
Material
research
Physics
Chemistry
Biology
EPR provides detection
and study of radicals
(R), systems with
unpaired electron spins
Polymers
Fullerenes
Glasses
Corrosion
Magnetic susceptibility
Semiconductors
Defects in crystals
Quantum dots
High T
superconductors
Oxidation and reduction processes
Biradicals and triplet state molecules
Reaction kinetics
Structure, dynamics, and reactions
of polymers
Photosynthesis
Protein labeling
Enzymatic reactions
Metallo proteins
Control of irradiated
food
Free radicals in living
tissue
Radical-initiated
carcinogenesis
Oxygen concentration measurement
Spin trapping of
short-lived radicals
14
Aplicación de EPR en Biología, Bioquímica y Medicina
Qué compuestos pueden ser estudiados por EPR ?
Sistemas paramagnéticos con electrones no emparejados, S ≠ O
1. Transition Metals: CuII,NiI,III,CoII,FeIII,MnII/III/IV,VIV,MoV, WV
2. Protein Side Chain Radicals (Tyr•,Trp•,Gly•,Cys•)
3. Radical states of Cofactors (Semiquinones, Flavins ...)
4. Inorganic Radicals (NO•,O2, O2•-, HO•....)
5. Transient Species in Light Driven Processes
...but also
1. Spin Traps can be used to Quench Short-Lived Radicals
2. Spin Labels can be attached to Proteins, Nucleic Acids, ... to
study their Structure and Dynamics (SDL) 15
Información por EPR
1. Is the substance paramagnetic ?
Note: some Integer Spin Systems are EPR silent !
2. Which type of paramagnet is present ?
... Fingerprinting! Metal, Organic Radical, Interacting systems
3. How much paramagnet is present ?
... Quantitation!
4. Information about geometric and electronic structure of paramagnet
5. Transition Metals - Information about type and number of ligands
6. Interacting Systems – Information about distances
7. Images in vivo
16
g-value
Flavin semiquinone, ubiquinone,
ascorbate …
2.0030 -
2.0050
Nitroxide spin labels and traps 2.0020 -
2.0090
sulfur radicals : RS-SR, RS-H 2.02 - 2.06
MoV (in aldehyde oxidase) 1.94
Cu2+ 2.0 - 2.4
Fe3+ (low spin) 1.4 - 3.1
Fe3+ (high spin) 2.0 - 10
g-valores para biológicamente importante
compuestos paramagnéticos
17
g valores (hasta 18) en sistemas complejos
18
40 60 80 100 120 140 160 180
14.8
g=17.5
Magnetic Field [mT]
15.0 10.7 8.34 6.82 5.77 5.00 4.41 3.94
g- Value
30 40 50 60 70 80 90
A Bg=9.7
Magnetic Field [mT]
18.8 15.0 12.5 10.7 9.38 8.34
g- Value
A. EPR spectrum of sulfite reductase from A. fulgidus. B. Low-field spectrum of sulfite
reductase. EPR conditions: 20.5 mg ml-1 sulfite reductase as isolated in 50 mM potassium
phosphate pH 7.0, 5 % glycerol, under exclusion of dioxygen; microwave frequency,
9.377 GHz; microwave power, 2 mW; modulation amplitude, 1 mT; temperature, 10 K
Recordar: No medimos espectros de absorción verdaderos,
medimos espectros modulados
Field modulation – Absorption vs 1st derivative
19
Presentación de Espectros EPR
Presentation of EPR Spectra
1000 2000 3000 4000 5000
Magnetic Field (Gauss)
2nd
1st
Absorption
The Magnetic Field is
usually measured in Gauss (G)
units. The SI unit, however, is
the Tesla (T) !
1T = 10 000 G
1 mT= 10 G
Typical Resonance Field
Bres~3000 G=0.3T
20
Importante: Cryotechnologia - nitrógeno líquido y
helio líquido - Variable Temperature ($$$$)
Beinert & Sands, BBRC, 1966 21
Relaxation D.J.E. Ingram (1969) Biological and Biochemical Applications of Electron Spin Resonance,
A. Hilger Ltd., London
22
Linewidth P.F. Knowles, D. Marsh, H.W.E. Rattle, Magnetic Resonance of Biomolecules, Wiley, 1976
23
If a system exists in a certain
energy state for only a short
time duration, then the
energy of the state is not well
defined. The possible energy
range, ΔE, is related to its
lifetime, τ, by the relation
ΔE x τ ≈ h;
h x ν = gßH;
Δ H ≈ h/gß x Δ ν = h/gß x 1/τ
Relaxation – Line Width - Heisenberg D.J.E. Ingram (1969) Biological and Biochemical Applications of Electron Spin Resonance
∆E x ∆t h/2π
∆p x ∆x h/2π
∆ν = 1/2π x τ
24
Relaxation Evolution of a spin system is described by Bloch equations:
T1 spin-lattice or longitudinal relaxation time
T2 spin-spin or traversal relaxation time
When properly integrated, the Bloch equations will yield the X', Y', and Z
components of magnetization as a function of time.
Stationary solution in rotating frame gives a Lorentzian line 2
0
2
2
2
2
)(1
1)(
HHT
THF
p
))(2
1exp(
2)( 2
0
2
2
22 HHTT
HF p
Gaussian line = inhomogeneous broadening
EPR linewidth: HkT
e '
2
1
12
'
2 2
111
TTT
)/(1076.1 7 Gsrade
k=1 Lorentzian
2lnpk Gaussian
Mx’, My’ Mz – magnetization components in the
rotating frame
0=eH0 – the Larmor Frequency
25
Relaxation-Time Determination from Continuous-Microwave
Saturation of EPR Spectra A Lund, E Sagstuen, A Sanderud, Jmaruani, Radiation Research (2009), 172, 753-760
26
Recordar: geff vs ge (bound electrons)
In reality resonance does NOT always occur at the same field because
bound electrons also carry some Orbital ANGULAR MOMENTUM in
addition to their SPIN ANGULAR MOMENTUM.
nucleus
electron Additional Magnetic Moment mL l=r x p
Modification of Resonance Condition:
E=hnB(me+mL)=b|B| geff
MOLECULAR Quantity=ge+g
1000 2000 3000 4000 5000
Magnetic Field (G)
g>0 g<0
27
Recordar: Anisotropy of g
Fe x
y z
The relative orientation of B
and m = me+mL matters a lot !
Consider three extreme cases:
hn=-Bxmx=bBxgx
B
hn=-Bymy=bBygy
B
hn=-Bzmz=bBzgz
B
Thus, g becomes anisotropic: the „g-Tensor“
28
Recordar: Anisotropy of g and A En Soluciones Congeladas uno ha orientado al azar Moléculas y así nos tenemos que
integrar sobre todas las orientaciones posibles!
In Frozen Solutions one has Randomly Oriented Molecules, thus we have to
integrate over all possible orientations!
2000 3000 4000 5000
Magnetic Field (Gauss)
... ...
gz
gy
gx
Assume gz>gy>gx „Powder Pattern“
29
2000 3000 4000 5000
gmin
gmid
Magnetic Field (G)
gmax
RHOMBIC
gxgygz
AXIAL
gx=gy(=g)gz (=g||)
ISOTROPIC
gx=gy=gz
2000 3000 4000 5000
giso
Magnetic Field (G)
2000 3000 4000 5000
gII
g
g
Magnetic Field (G)
gII
„Bio Dialect“ for Powder Patterns
30
Recordar: Hyperfine Interaction
Some Nuclei behave like Little Bar Magnets (NMR Spectroscopy).
• The condition is that the nuclei have a Non-zero nuclear Spin I.
(i.e. 14,15N, 17O, 19F, 63,65Cu, 61Ni, 57Fe, 95Mo...)
• The Magnetic Interaction between the Nuclei and the Unpaired Electrons is called
Hyperfine Interaction (Symbol A)
• The Hyperfine Interaction leads to a Splitting of EPR Lines
No interaction Zeeman Interaction Hyperfine Interaction
|>
|>
|>
|>
Selection Rule:
The Nuclear Spin must not
change in an EPR Transition
gbB
A/2
A/2
Electron Spin
Nuclear Spin
Two Transitions with Different Energies
31
EPR Spectrum with Hyperfine Structure
1000 2000 3000 4000 5000
Amax
/bgmaxA
mid/bg
mid
Amin
/bgmin
gmin
gmid
Magnetic Field (G)
gmax
Nucleus with Spin I:
2I+1 Lines
Splitting Depends
on the Orientation
A is different for
each g-direction
„A-Tensor“
32
Equivalent Nuclei
In the case of n equivalent Nuclei with Spin I one obtains
a Hyperfine Pattern with 2nI+1 Lines and a Binomial
Intensity Distribution
I=1/2
n=1 1:1
n=2 1:2:1
n=3 1:2:2:1
n=4 1:2:3:2:1
I=1
1:1:1
1:2:3:2:1
1:2:3:4:3:2:1
1:2:3:4:5:4:3:2:1
33
Hyperfine Pattern: Naphthalene Radical Anion
a=4.9 Gauss
ab=1.83 Gauss
4 equivalent H = 1:2:3:2:1
(Organic radicals almost always have g-shifts
which are very close to the free electron g-
value ge=2.002319...) 34
Los elementos/metales de vida www.webelements.com
Ca: 1.2 kg
K: 150 g
Na: 70 g
Mg: 20-30 g
Essential
Transition Metals
Fe: 4.5 g
Zn: 2.3 g
Cu: 72 mg
Mo: 9 mg
H He
Li Be B C N O F Ne
Na Mg Al Si P S Cl Ar
K Ca Sc Ti V Cr Mn Fe Co Ni Cu Zn Ga Ge As Se Br Kr
Rb Sr Y Zr Nb Mo Tc Ru Rh Pd Ag Cd In Sn Sb Te I Xe
Cs Ba La Hf Ta W Re Os Ir Pt Au Hg Tl Pb Bi Po At Rn
Los metales de transición están bajo el
control estricto dentro de la célula
viva; por lo general ligado a proteínas,
péptidos u otras moléculas.
En la QUÍMICA: COMPLEJO DE
METAL DE TRANSICIÓN
36
Metales y Vida:
la Química de Coordinación de la Naturaleza
“El uso de metales para tratar dolencias humanas se remonta al
menos al quinto siglo a. de J.C., y el estudio y la imitación de
metales en la biología son un sujeto vibrante hoy”
Stephen Lippard, J Am Chem Soc (2010), 132, 141689-14693
37 B. Rosenberg et al., (1965) Nature, 205, 698 - 699
Metales en Medicina – Aplicaciones “Uno de los desafíos de diseñar medicinas basadas en el metal es equilibrar la
toxicidad potencial de una formulación activa con el impacto positivo sustancial
de estos recursos terapéuticos y diagnósticos cada vez más comunes” K.H. Thompson, C. Orvig (2003) Science 300, 936-939
38
Principios básicos de un complejo de la proteína metálico Chem. Rev. 1996, 96, 2239-2314 (1996) RH Holm, P Kennepohl, E I Solomon, Structural and
Functional Aspects of Metal Sites in Biology
Prot-L| M - +
strong attraction
--------
39
Plastocyanin, un complejo de
Cobre : Cu(II) + e- Cu(I) → ←
Sirohemes (Fe+2,
Fe-S cluster)
OO
NH
NH
NH
NH
O
O
O
O
O
O
O OOO
O
O
O
O
D
A B
C
Tetrapyrroles - Ligantes versátil en Biología
N & S
Cycles
Photosynthesis
(Bacterio)- Chlorophylls (Mg+2)
Met biosynthesis
VitB12 (Co+2)
O2 Respiration
F430 (Ni+2)
Methanogenesis
Hemes (Fe+2)
41
Molybdopterin, a Dithiolene Ligand (binds both Mo and W) JOURNAL of BIOLOGICAL CHEMISTRY (2009) Vol. 284, p. e10, N Kresge, R D Simoni, R L Hill: The
Discovery and Characterization of Molybdopterin - the Work of K. V. Rajagopalan
Mo-S-Cu Cluster in CO Dehydrogenase from Oligotropha carboxidovorans :
CO + H2O → CO2 + 2 H+ + 2e- H Dobbek et al., Proceedings National Academy of Sciences/USA, 99, 15971-15976 ( 2002) 42
Recordar: La Geometría es importante
3
4
5
6
Trigonal Trigonal pyramidal T-shape
Square planar Tetrahedral
Square pyramidal Trigonal bipyramidal
Octahedral
43
La Geometría es importante: Proteínas de Hierro
EI Solomon, et al. (2000), Chem. Rev., 100, 235–350
Rubredoxin
3,4-Protocatechoate
Dioxygenase
Tyrosine
Hydroxylase
Lipoxygenase
Tetrahedron Trigonal
Bipyramide
Tetragonal
Pyramide Octahedron
44
Historia 1:El descubrimiento de un nuevo Centro de Cu D KEILIN, T MANN, Nature, 143, 23-24 (1939)
B G MALMSTRÖM, R MOSBACH, T VÄNNGÅRD, Nature, 183, 321-322 (1959)
1939: Laccase, a Blue Copper Protein from the Latex of Rhus succedanea
1959: An Electron Spin Resonance Study of the State of Copper in Fungal
Laccase
Type 1 Blue Copper Electron Transfer Center Spectroscopic
Methods in Bioinorganic Chemistry: Blue to Green to Red Copper Sites.
E I Solomon, Inorg. Chem., 45, 8012-8025 (2006)
45