V

Mn Fe

Co

Ni

C

Cu

Mo W

N

O S

Bioinorganic CW EPR Spectra Stefan Stoll University of Washington, Seattle 4th Penn State Bioinorganic Workshop June 5, 2016

Goals 1. Improve understanding of CW EPR a. derivative vs absorption b. “powder” spectra 2. Learn what you can learn from CW EPR

Contents 1. Overview 2. Basics and examples a. Ni, Cu, Co b. Fe, Mn c. mixed-metal d. organic radicals 3. Summary

Disclaimer: This is not a comprehensive survey of CW EPR spectra

EPR survival kit Textbooks

Simulation software

EasySpin

Bioinorganic EPR Periodic Table organic radicals

H Li

Be

transition metal complexes & clusters

Na Mg K

Ca

Sc

Ti

V

Rb

Sr

Y

Zr Nb Mo Tc

N

O

F

Ne

Al

Si

P

S

Cl

Ar

Cu

Zn Ga Ge As

Se

Br

Kr

Ru

Rh

Pd

Ag

Cd

In

Sn Sb

Te

I

Xe

Re Os

Ir

Pt Au Hg

Tl

Pb

Po

Fr

Rf Db Sg Bh Hs Mt Ds Rg Cn

La Ce

C

Ni

Hf

Lr

W

B

Co

Cs Ba Lu Ra

Ta

Cr Mn Fe

He

Fl

Bi

At Rn

Lv

Pr Nd Pm Sm Eu Gd Tb Dy Ho Er Tm Yb

Ac Th Pa

U

Np Pu Am Cm Bk

Cf

Es Fm Md No abundant in living organisms EPR-active transition metal complexes EPR-active radicals

Most common metals and oxidation states half filled

less than half filled 3d1

3d2

3d3

1/2

1

3/2

V(IV)

3d4

3d5

2 0

5/2 1/2

V(I) V(II) V(III) Cr(IV)

more than half filled 3d6

3d7

3d8

3d9

3d10

1

1/2

0

Cu(I)

2 0

3/2 1/2

Cr(I)

Mn(I)

Fe(I)

Co(I)

Ni(I)

Cr(II)

Mn(II)

Fe(II)

Co(II)

Ni(II)

Cu(II)

Cr(III)

Mn(III)

Fe(III)

Co(III)

Ni(III)

Cu(III)

Mn(IV)

Fe(IV)

Co(IV)

Ni(IV) half-integer spin (easy to observe) integer spin (difficult or silent)

Typical sample: Frozen aqueous solution of a protein B0

Frozen solution = random uniform distribution of static orientations of protein molecules (like a dilute powder)

static magn. field B1 mw magn. field

Lab-fixed frame - z(lab): static field B0 - x(lab): oscillating microwave field B1

proteins

Random relative orientation

Protein-fixed frame - aligned with local symmetry z y

z(lab)

x y(lab) x(lab)

Ni Cu Co Fe Mn

Nickel(I) Ni(I) Ni(II)

*

3d9 S = 1/2 3d8 S = 1 high-spin S = 0 low-spin (nonmagnetic)

61Ni

(I = 3/2) 1%

z y CW EPR

𝑩𝑩0

x z y

abs

g = 2.56, 2.10, 2.01; mwFreq = 9.5 GHz

y z

C

Ni Cu Co Fe Mn

Nickel(I)

*

Acetyl-coenzyme A synthase (ACS)

C

Ared*

Nip(I) Nid(II) Biochem. 2010 49 7516

Ni-substituted azurin (NiAz)

d𝑥𝑥 2−𝑦𝑦2 trigonal planar

𝑔𝑔∥ = 2.56

𝑔𝑔⊥ = 2.10

Ni(I) Inorg. Chem. 2015 54 7959

Ni Cu Co Fe Mn

Nickel(I)

*

Methyl-coenzyme M reductase (MCR) F430 = Ni hydrocorphinate cofactor

C

g = 2.25, 2.08, 2.06

Science 2016 352 953

Ni superoxide dismutase (NiSOD)

gx = 2.30

quantitation: 50% Ni(I) 50% ox.

gy = 2.23 5-coord. d𝑧𝑧 2

gz = 2.01 14N

Ni(I)

hf splitting! Biochemistry 2015 54 1016

Ni Cu Co Fe Mn

Copper(II)

*

Cu(II) 3d9 S = 1/2 Cu(I) 3d10 S = 0 (EPR silent)

CW EPR

Azz

63Cu

(I = 3/2, 4 spin states) 69% 65Cu (I = 3/2, 4 spin states) 31% z = parallel = ∥

Azz

Azz

abs

C

x,y = perpendicular = ⊥

sum

𝑚𝑚𝐼𝐼 𝑚𝑚𝐼𝐼 𝑚𝑚𝐼𝐼 𝑚𝑚𝐼𝐼 g = 2.06 2.06 2.25; A = 50 50 400 MHz; mwFreq = 9.5 GHz

= −3/2 = −1/2 = +1/2 = +3/2

25% 25% 25% 25%

Copper(II) Cu(II) proteins

Ni Cu Co Fe Mn * Peisach/Blumberg plot gz and Az are anticorrelated

transferrin

plastocyanin

azurin

stellacyanin

Arch. Biochem. Biophys. 1974 165 691 J. Magn. Reson. 2013 236 7

JACS 1984 106 5324

𝑑𝑑𝑥𝑥 2−𝑦𝑦2 vs. 𝑑𝑑𝑧𝑧 2 character delocalization onto ligands coordination geometry

C

Ni Cu Co Fe Mn

Copper: clusters Cu(II)-Cu(II): Cu(II) acetate antiferromagnetic coupling nonmagnetic (S = 0)

* Tyrosinase: CuICuII cluster

C

𝑑𝑑𝑥𝑥 2 −𝑦𝑦2

no EPR spectrum Inorg. Chem. 2011 50 6229

𝑑𝑑𝑧𝑧 2 admix. trig. bipy. Chem. Rev. 2014 114 4366

Nitrous oxide reductase: CuZ = Cu4S “1-hole”: 3CuICuII S = 1/2 “2-hole”: 2CuI2CuII S = 0

1-hole deloc., 𝑑𝑑𝑥𝑥 2−𝑦𝑦2 2.152

2nd deriv Chem. Sci. 2015 6 5670 Biochemistry 2000 39 12753

2.042

Ni Cu Co Fe Mn

Cobalt(II) Co(II)

3d7 S = 3/2 (high spin) S = 1/2 (low spin)

59Co

(I = 7/2, 8 spin states) 100%

Co(II) porphyrin: dioxygen binding

hfc resolved

without O2 Angew. Chem. 2008 47 2600

hfc discernible

O2 bound

g value changed → change of coordination geometry/environment

* C

Ni Cu Co Fe Mn

Iron: mono-nuclear complexes Fe(III) Fe(II)

3d5 (S = 1/2, low-spin) (S = 5/2, high-spin) 3d6 (S = 2 high-spin)

*

57Fe

2.2% I = 1/2 (2 spin states)

Cytochrome P450cam: Fe(III) 8

+cam

1.98 high-spin

4

2.26 2.45

1.91

low-spin 1.8

-cam

2.41 2.24

high-spin Fe(III) g-values are “effective” for lowest 2 states of 6. (take into account zero-field splittings)

low-spin 1.96 Biochem. 1976 15 5399 Biochem. 1980 19 3590

C

Ni Cu Co Fe Mn

Iron-sulfur centers 1Fe

Fe2+ Fe3+

S=2 S = 5/2

2Fe2S

Fe2+Fe3+ 2Fe3+

S = 1/2 S=0

3Fe4S

2Fe2.5+Fe3+ 3Fe3+

S=2 S = 1/2

4Fe4S

others

2Fe2+2Fe2.5+ 4Fe2.5+ 2Fe2.5+2Fe3+

S = 1/2 S=0 S = 1/2

C

4Fe4S Radical SAM enzymes

JACS 2010 132 2037

-SAM

+SAM

4Fe4S + Fe-heme (siroheme) 4Fe4S + 2Fe (hydrogenase) 8Fe7S P-cluster (nitrogenase)

Chem. Rev. 2014 114 4366

*

2Fe2S Ferredoxins et al.

JACS 2002 124 3143

Ni Cu Co Fe Mn

Manganese(II) Mn(II) 3d5 S = 5/2 (high-spin, 6 spin states)

CW EPR 𝑚𝑚𝐼𝐼 = −5/2 −3/2 −1/2 +1/2 +3/2 +5/2

abs

*

55Mn

100% (I = 5/2, 6 spin states)

C

• isotropic g and A insensitive • zero-field splitting sensitive to coordination environment central transition −1/2 ↔ +1/2 +1/2 ↔ +3/2 −1/2 ↔ −3/2 +3/2 ↔ +5/2 −3/2 ↔ −5/2 −1/2 ↔ +1/2

g = 2, A = -240 MHz, D = 200 MHz, Dstrain = 150 MHz; mwFreq = 9.5 GHz

allowed Δ𝑚𝑚𝐼𝐼 = 0

forbidden Δ𝑚𝑚𝐼𝐼 = ±1

Review on Mn(II) EPR: Appl. Magn. Reson. 2010 37 229

Ni Cu Co Fe Mn

Manganese(II)

*

Comparison of coordination geometries GHz

C concanavalin-A (octahedral) bacterial RC (low-symm 6-coord.) MnSOD (trigonal bipyram. 5-coord.) BBA 2010 1804 308

Oxalate decarboxylase

J.Phys.Chem.B 2007 111 5043

Manganese(III) Oxalate decarboxylase

Mn(III)

parallel-mode EPR 𝐵𝐵1 ⊥ 𝐵𝐵0

3d4

(S = 2, high-spin)

Ni Cu Co Fe Mn *

Mn(III) myoglobin

C

𝐵𝐵1 ⊥ 𝐵𝐵0

𝐵𝐵1 ⊥ 𝐵𝐵0

Biochem. 2016 55 429

Mn superoxide dismutase

JIBC 2008 102 781

3𝑑𝑑𝑧𝑧 2 empty mixture

3𝑑𝑑𝑥𝑥 2−𝑦𝑦2 empty

JACS 2011 133 20878

Ni Cu Co Fe Mn

Manganese: Mn2 clusters Mn2(II,II) Mn2(II,III) Mn2(III,III) Mn2(III,IV)

S=0 S = 1/2 S=0 S = 1/2

*

Mn catalase (II,III)

C (III,IV)

anti-ferromagnetic coupling

(III,IV)

6x6 = 36 hf lines

Inorg. Chem. 1994 33 382 JPCB 2003 107 1242

Manganese: Mn4 cluster

Ni Cu Co Fe Mn * C

Photosystem II

“multiline signal”

Ni Cu Co Fe Mn

Substrate, cofactor and protein radicals Protein radicals tyrosyl, typtophanyl, cysteinyl, glycyl Cofactor radicals semiquinones, flavins, 5’-dA, … Substrate radicals many!

* S = 1/2 many hfc

Photosystem II x

Electron Paramagn. Reson. 2004 19 174

Review high-field EPR bioorganic radicals: Electron Paramagn. Reson. 2011 22 107

high-field EPR resolves g-tensor

y

z

C

Ni Cu Co Fe Mn

Mixed-element clusters Mn-Fe: Ribonucleotide reductase (Ic)

* Fe-radical: Cytochrome P450 cmpd I 2.00 effective g values

Stot = 1/2 Mn(III) S = 2 Fe(III) S = 5/2

Stot = 1/2 Fe(IV)=O S = 1 radical S = 1/2

1.72 1.61

CPO Stot = 1/2 Mn(II) S = 5/2 Fe(II) S = 2

2.00 1.96 1.86

CYP119-I Mn Science 2007 316 1188 J.Biol.Chem. 2009 284 4555 Biochem. 2013 52 6424

Fe

Science 2010 330 933

C

Ni Cu Co Fe Mn

Mixed-element clusters

* C

CO dehydrogenase (CODH): Mo-Cu CO-reduced wt

95,97Mo

Ag(I)

(I = 5/2) 25%

Mo(VI) 3d0 S = 0 Mo(V) 3d1 S = 1/2

g = 2.0043, 1.9595, 1.9540 A = 82.0, 78.9, 81.9 MHz

JACS 2011 133 12934

107,109Ag

I = 1/2

Mixed-element clusters Cu-Fe: Cytochrome c oxidase Cu-Cu

Ni Cu Co Fe Mn Cytochrome bo3 Fe(III, S=5/2)-Cu(II, S=1/2)

perp. mode paral. mode

heme-copper Cu-Fe 4.5 Å Biochem 2002 41 2288

* C

Summary Paramagnetic species in bioinorganic systems V

Mn Fe

Co

Ni

Cu

C

Mo W

N

O S

and many combinations thereof!

CW EPR workflow CW EPR spectra

EPR parameters (S, g, D/E, metal & ligand A)

Key CW EPR concepts for novices • derivative vs. absorption • powder spectra / orientation selection

Structure

10 Things you can learn from CW EPR spectra 1. spin concentration (µM) 2. identity of paramagnetic center (Cu, FeS, radical) 3. oxidation states (I, II, III, IV) 4. spin states (high-spin, low-spin) 5. nature of half-occupied orbitals (𝑑𝑑𝑧𝑧 2 etc) 6. coordination geometry (octahedral,…) 7. ligand binding 8. nature of ligands 9. spin delocalization onto ligands 10. cluster valence (de)localization