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

peter.kroneck@uni-konstanz.de

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

I=1/2, 2I+1=2

I=1, 2I+1=3

35

Nitrogen Hyperfine – 14N vs 15N

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) → ←

Proteína Ligantes – residuos del aminoácido

N O S

His

Lys

Tyr

Glu(+Asp)

Ser

Cys

Met

40

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