Pregunta : Tiene una solución de 1.0 mM de [Cu(NH3)4]2 + !

Dibuje los espectros EPR medidos en 300 y 100 K !

1

Pregunta: Bajo la aceptación de una geometría cuadrado planar

de Cu(II) dónde es gI (g-perpendicular) y dónde g║(g-parallel) ?

Pregunta : Explica Hyperfine Pattern (alfa protones) de

Naphthalene radical anion ! („Stick diagram“)

Selected Metals: Copper Proteins

Concept of Malkin and Malmström R Malkin, BG Malmström, Advances Enzymology, 33,177-244 (1970)

Classification of Cu sites according to UV/VIS

and EPR Properties: Type 1 (blue; mononuclear),

Type 2 (non-blue; mononuclear), Type 3 (EPR-

silent; dinuclear)

Blumberg-Peisach Plot (gII vs AII) W E Blumberg, J Peisach, in Probes of Structure and Function of Macromolecules and

Membranes, ed. B Chance, Academic Press, New York, 1971, vol. 2, pp. 215–229

J Peisach, WE Blumberg, ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS, 165, 691-

708 (1974)

2

Blumberg-Peisach Plot - gII vs AII – Cu Proteins

3

Cu A, in Cytochrome c

Oxidase and Nitrous

Oxide Reductase

EPR of square-planar CuCl42-

Solomon et al., Chem. Rev. (1992), 92, 521-542

4

Propiedades Únicas de Cu Azul (tipo 1)

See Blumberg-Peisach Plot

Hans Freeman, 1978

5

Bioengineering: tipo 1 Cu → tipo 2 Cu

Handbook of Metalloproteins (A Messerschmidt, T L Poulos, K Wieghardt, R Huber, eds), Wiley, 2001

EPR (a) EPR (c) EPR (d)

Type 1 Cu in

Azurin

Cys112

6

Bioengineering of Electron Transfer Centers : tipo 1 Cu → tipo

CopperA (dinuclear mixed-valent) Work of G Canters/Leiden/NL and Y Lu, Urbana/USA; MG Savelieff, Y Lu, J Biol Inorg Chem, 15,

967-976 (2010)

Plastocyanin/Photosynthesis Cytochrome c Oxidase/Respiration

Nitrous Oxide Reductase/Denitrification 7

Cu Hyperfine Structure – Nitrous Oxide Reductase Coyle, Zumft, Kroneck, Körner, Jakob (1985) Eur. J. Biochem., 153, 459; Neese, Zumft,

Antholine, Kroneck (1996) J. Am. Chem. Soc., 118, 8692; Pomowski, Zumft, Kroneck, Einsle

(2011), Nature, 477, 234

2600 2800 3000 3200 3400

Magnetic Field (G)

CuA ICu=3/2

7-Lines Observed

#Lines=2nI+1

n=2

2x2x3/2+1=7

1:2:3:4:3:2:1

Expected and Observed

2 Equivalent

Cu predicted

8

7-line EPR Spectrum of CuA (2nd derivative)

One electron delocalized over two Cu nuclei

Note the short (metallic) Cu-Cu distance ≈ 2.5 Å

9

Galactose Oxidase - una enzima radical de Cu covalent linkage between Tyr272 and Cys228

10

PDB:1GOF

Galactose Oxidase - una enzima radical de Cobre

RCH2OH + O2 RCHO + H2O2

GalOx

EPR

EPR

11

Galactose Oxidase - Mechanism Que, Tolman (2008) NATURE 455, 333

12

Historia 2:El descubrimiento de un nuevo Centro de Fe IDENTIFICATION BY ISOTOPIC SUBSTITUTION OF THE EPR SIGNAL AT g = 1.94 IN A NON-HEME

IRON PROTEIN FROM AZOTOBACTER, Y I SHETHNA, P W WILSON, R E HANSEN, H BEINERT,

Proceedings National Academy of Science/USA (1964), 52, 1263-1271

EPR spectra of Fe56 and

Fe57 iron proteins

superimposed. The dotted

curve represents a

computed curve for the

Fe57 protein, which was

obtained from the curve of

the Fe57 protein, assuming

a hyperfine splitting of 22

G and a final enrichment

of 65% for Fe57 , IFe57 = 1/2.

13

Selected Metals: Iron-Sulfur/FeS Proteins Sulfide (S2-, inorganic/labile) & CyS- (organic/stable)

Spinach [2Fe-2S] Ferredoxin PDB 1A70 Electron Transfer Protein

14

Redox states of [2Fe-2S]-Cluster

ox state mixed-valent state

high-spin Fe high-spin Fe

S = 0 ; diamagnetic S = 1/2 ; paramagnetic; unpaired

e- delocalized over both Fe centers

delocalized

discrete, Fe(II) and Fe(III) centers

•antiferromagnetic coupling between both Fe centers

•[Fe2S2]+ with S = 9/2 (2x Fe(2.5))

REDOX of [2Fe-2S] center

15

Redox states of [4Fe-4S]-Cluster

HiPIP-type Ferredoxin-type

S = ½ S = 0 S = ½

paramagnetic diamagnetic paramagnetic

• Redox potentials: +50 to +450 mV (HiPIP)

-650 to 0 mV (Ferredoxin)

• Higher potentials of HiPIPs: different redox couples

REDOX [4Fe-4S] center antiferromagnetically coupled Fe2S2-units

16

Recordar: Fe-S Centers – EPR Finger Printing

300 320 340 360 380 400

Magnetic field [mT]

[4Fe-4S]3+/2+

HiPIP

[4Fe-4S]2+/+

[2Fe-2S]2+/+ Ferredoxin

[2Fe-2S]2+/+ Rieske

oxidized gav=2.06

[3Fe-4S]+/0

oxidized gav=2.01

reduced gav=1.96

reduced gav=1.96

reduced gav=1.91

gy=1.91

gx=1.79

gz=2.02

gz=2.05

gy=1.95

gx=1.89

gz=2.06 gy=1.92

gx=1.88

gz=2.12 gx,y=2.04

gz=2.02

gx,y=2.00

17

Heme Iron – high vs low spin Fe(III) Hagedoorn, de Geus, Hagen (2002) FEBS J. 269, 4905

18

BRAUTIGAN, FEINBERG, HOFFMAN, MARGOLIASH, PEISACH, BLUMBERG

J. Biol. Chem. (1977) 252, 574

19

Selected Metals: Molybdenum/Tungsten Proteins

RC Bray, LS Meriwether, Nature, 212, 467-469 (1966)

R Hille, Archives of Biochemistry and Biophysics, 433 107–116 (2005)

Molybdenum EPR signal of Xanthine Oxidase

(XO) reacted with formaldehyde. Bottom trace:

XO, nat. abundance; central trace : Mo-95

enriched XO; upper trace: central trace, enlarged 20

21

????????????????

You figure out the

geometry of the

Mo(V) site !

Hyperfine splittings

from 95Mo and

Hydrogen/Substrate.

EPR Prediction: Acetylene Hydratase is a Tungsten, Iron-Sulfur Enzyme

RU Meckenstock, R Krieger, S Ensign, PMH Kroneck, B Schink, Eur J Biochem, 264, 176–82 (1999)

3300 3400 3500 3600 3700-20

-15

-10

-5

0

5

10

15

20

25

30

A

B

1.920

1.9352.048

Re

lative

in

tensity

Magnetic field [G]

-6

-4

-2

0

2

2,15 2,1 2,05 2 1,95 1,9 1,85

2.005

2.048

C

Rela

tive in

ten

sit

y

2.015

g value

320 330 340 350 360 370 380

Magnetic field [mT]

Substrate

Binding

[4Fe-4S] < 20 K, Electron Transfer

W catalytic site, > 20 K

C2H2 + H2O → [H2C=C(OH)H] → CH3CHO

22

3D Structure of Tungsten Acetylene Hydratase GB Seiffert, MG Ullmann, A Messerschmidt, B Schink, PMH Kroneck,O Einsle, Proc Natl Acad Sci USA,

104, 3073–77 (2007).

PDB code: 2E7Z

23

Tyrosyl Radicals in Biology

Tyrosine:

• Weak O-H Bond (87 kcal/mol) due

to stabilization of the radical

• pKa=10

• Redoxpotential=650-1100 mV ( vs

NHE)

Tyrosyl Rdical Enzymes:

• Ribonucleotide Reductase

• Photosystem II

• Galactose Oxidase

• Catalase

• Prostaglandin H Synthase

24

EPR Spectra of Tyrosyl Radicals

3240 3260 3280 3300 3320 3340

Magnetic Field (G)

87100 87200 87300 87400 87500

2.0091

2.0067

2.0045

2.0023

Magnetic Field (G)

X-Band

9.2 GHz High Field

245 GHz

Large variation in gmax values reflects different protein

environments and carries electronic structure information

E. Coli

RNR

Mouse

RNR

25

H-Bonding in Tyrosyl Radicals

out-of-plane

lone pair

in-plane

lone pair

De1 De2

H-bond

26

H-Bonding Effects on gmax

O

O

gmax

LPop

LPip

• Rotation around the C-O bond direction creates

a large angular momentum along the C-O bond

• gmax is along C-O

• Rotation is the more effective the smaller the

energy gap

• H-bonding quenches angular momentum

1.6 1.7 1.8 1.9 2.0 2.1

8850

8900

8950

9000

9050

9100

9150

H-Bond distance (A)

Exci

tation E

nerg

y (

cm-1)

2.0064

2.0067

2.0070

gm

ax

27

Outlook – EPR técnicas avanzadas

28

S> 1/2 systems: Triplet states ; Transition Metal Ions (e.g. h.s. Fe(III) and

Fe(II); Heme Fe; Mn(II), Mn(III), Mn(IV))

Wertz, Bolton (1986) Electron Spin Resonance, Elementary Theory and Practical

Applications, Chapmann and Hall; Cammack, Cooper (1993) Meth. Enzymol.

227, 353.

Advanced techniques: ENDOR; Pulse EPR/ENDOR; Spin Labeling;

Distance Measurements; Imaging

Mims, Proc. R. Soc. Lond. A (1965) 283, 452; Hoffman (2003) PNAS 100, 3575;

Murphy, Farley (2006) Chem. Soc. Rev. 35, 249; Eaton, Eaton (2012) J. Magn.

Reson., 223,151; Van Doorslaer, Desmet (2008) Meth. Enzymol. 437, 287;

Roser, Schmidt, Drescher, Summerer (2016) Org. Biomol. Chem. 14, 5468.

Adenosine-5‘-phosphate Reductase (PDB code: 1JNR) G Fritz, A Roth, A Schiffer, T Büchert, K Bourenkov, HD Bartunik, H Huber, KO Stetter, PMH

Kroneck, U Ermler, Proc Natl Acad Sci USA, 99, 1836-1841 (2002)

29

Interaction between the two [4Fe-4S] - Centers

G Fritz, T Büchert, PMH Kroneck, J Biol Chem, 277, 26066-26073 (2002)

10.8 Ǻ

310 320 330 340 350 360 370 380

Magnetic field [mT]

2.078 1.941 1.902 2.066 2.045 1.976 1.938 1.890

310 320 330 340 350 360 370 380

Magnetic field [mT]

I II I II

Eo‘ = - 60 mV Eo‘ = - 520 mV

[+] [2+] [+] [+]

15.4 Å9.8 Å

R

II I

30

ENDOR (Electron Nuclear DOuble Resonance) provides missing information

3390 3400 3410 3420

g = 2.0026

Magnetic Field / Gauss

8 10 12 14 16 18 20 22

Mutant

12I

12II2II

7II

7I

2I

1'

7II2II

2I

12I

12II

17

17

18

18

7I2'

2

Frequency / MHz

1

WT

H

P700 radical cation

(Chlorophyll a dimer)

EPR

ENDOR

31

Applied for many biological systems with small hyperfine coupling

constants which are not resolved in conventional CW EPR spectrum

3360 3370 3380 3390 3400 3410

Magnetic Field / Gauss

2 4 6 8 10 12 14 16 18 20 22 24 26 28

H = 14.3 MHz

Frequency / MHz

.1

2

NMR = n a/2

A1

A2

EPR ENDOR

Phenalenyl radical

Hyperfine enhancement effect

32

Spin labeling- Peptides and Proteins

R N

O

OH

N

HO

O

+DCC O

NO

R

OO

N O

Nitroxides are introduced into proteins as reporter groups to provide information

about local environment, overall tumbling rate of the protein or/and segmental

mobility, accessibility of the labeling site for polar/non-polar molecules, distance

measurements to other spin labels, co-factors, membrane surface….

Labeling of the hydroxyl group

NO

CH2SSO2CH3Protein SH + S CH2

NO

SProtein

MTSL spin label is cysteine specific.

SDSL = site directed spin labeling is introducing cysteines into the protein

molecule by point mutations with following MTSL labeling.

Cysteine mapping of the protein.

33

Modern pulse EPR techniques (DQC, DEER) can cancel out all interactions

resulting in an EPR spectrum except the dipole interaction in spin pairs

Example: spin labeled Gramicidin A

3/2 re D

dipolar, MHz,

2/][]Å[

102.53

4

MHzr

dipolarD

Å.930Interspin distance=

34 DEER = double electron electron resonance; DQC = double-quantum coherence

DQC-EPR ruler

G-C

C-G

A-U

G-C

C-G

U-A

G-C

A-U

U-A

G-C

G-C

C-G

C-G

U-A

A-U

C-G

G-C

C-G

G-C

U-A

G-C

U-A

U-A

C-G

(O*N) -U

5'

3'

U-(N*O)3'

5'

G-C

C-G

A-U

G-C

C-G

U-A

G-C

A-U

U-A

G-C

G-C

C-G

C-G

U-A

A-U

C-G

G-C

C-G

G-C

U-A

G-C

U-A

U-A

C-G

(O*N) -U

5'

3'

U-(N*O)3'

5'

10 20 7030 40 50 60 1009080

DQC RULER

Å

Location of mobile domains in a protein

complex using DQC-EPR

35

Oxygen Accessibility

W. L. Hubbell, H. S. Mchaourab, C. Altenbach, and M. A. Lietzow,

Structure 4, 779-783 (1996).

Oxygen accessibility and probe

mobility were measured as a

function of sequence number for

spin labels attached to T4

lysozyme (T4L) and cellular

retinol binding protein (CRBP).

The correlation between the two

parameters indicates that the

most mobile sites are also the

most oxygen accessible.

The repeat period of about 3.6

for T4L is consistent with the a-

helical structure of this segment

of the protein.

36

EPRI - See Video Center for EPR Imaging, The University of Chicago, USA

https://epri.uchicago.edu/page/epr-imaging

37

EPRI - EPR IMAGING

In Vivo Physiological Studies

38