Unveiling Nature. Electronic, Magnetic and Catalytical

Properties of Copper Proteins Dissected by Functional

Synthetic Systems

Max Planck Institute Polymer Research

Dr. rer. nat. Giorgio Zoppellaro

ACTIVOXY

Biological activation of O2

The biradical nature O2

EVOLUTION

O2

Mimicking Spectroscopic and Structural Properties of the Natural Systems

Elucidate Electronic and Magnetic Properties, Details of the Reaction Paths Mechanisms in the Natural Systems when those are Difficult to Unveil

Mimicking Functions, Reactivities and Selectivities

Target enzymes under investigation: copper oxidases/monoxygenase

Drugs Design

Green Chemistry

Sensors

Energy storage/conversion (Biof. Cells)

Cu c

onta

inin

g e

nzym

es t

hat

activate

O2

Fungal/Plant

Mammal Mammal gland

Mammal brain

Pituitary, heart

Mollusks

Animal serum

Plant

Fungal Most animals Mitochondria

Red-blood cells

Molds

Met. bacteria

1 Cu +

2 Cu

2 Cu

Type I

Type II Type III

His

Cys

Met

Glu,

Asp

Type II + III Cluster

Type III Type II /

Cu Cu

Cu

O

N

S

Types of Copper in proteins (nomenclature)

C112D mutant Cys replaced by aspartic acid

M121 replaced by isoleucine / leucine / phenyl-alanine G45 (glycine)

Becomes a ligand for Cu

112

G45

Galactose oxidase (0.17 nm) Dooley, D. M. Biochem. 2007, 46, 4606

Type II

Cathecol oxidase (0.18 nm) Krebs B. Nature Struct. Biol. 1998, 5, 1084−1090.

Type III met

Type I

Laccase Lintinus tigrinus (0.15 nm) Briganti F. BMC Struct. Biol. , 2007, 60

Type II, III

Tyr-Cys

Type I Type II

Peptidylglycine Hydroxylating Monooxygenase (0.14 nm) Prigge S. T. Science , 1997, 1300

In humans, PAM is the

target of drug design for

diseases ranging from

rheumatoid arthritis to

cancer.

broad substrate

specificity,

since peptides with

all 20 amino acid

amides have been

isolated

Zn2+

Multicopper oxidases

Evidence for Substrate Preorganization in the

Peptidylglycine α-Amidating Monooxygenase

Reaction Describing the Contribution of Ground

State Structure to Hydrogen Tunneling

Merkler D.J., J. Am. Chem. Soc., 2010, 132 (46), pp 16393–16402

Dopamine

beta

hydroxylase /

Parallelism

with PHM

The Raper-Mason Scheme (adapted from http://omlc.ogi.edu/spectra/melanin/melaninsynth.gif (Prota, 1988)). From Antje Kristina Biesemeier, PhD thesis, EBERHARD KARLS UNIVERSITÄT TÜBINGEN, 2010

Fe containing protein

• DBH requires ascorbate as a cofactor. It is the only enzyme involved in the synthesis of small-molecule neurotransmitters, which is membrane-bound.

Tyrosin hydroxylase

Tetrahydrobiopterin / O2

Di-hydrobiopterin

/ water

Ascorbic acid / O2

Decarboxylase

Phenylethanolamine

N-methyltransferase

S-adenosylmethionine

homocysteine

Type 0 copper centers (T0Cu, ARTIFICIALLY engineered from azurin mutants): two histidine

plus aspartic acid and leucine/isoleu/ phenylalanine. Set donors 2N/2O. They work as electron

transfer.

Type I copper centers (T1Cu, found in Nature) are characterized by a single copper atom

coordinated by two histidine residues and a cysteine In class I T1Cu proteins (amicyanin,

pseudoazurin, plastocyanin) other ligand is the sulfur of methionine. II T1Cu copper proteins

contains glutamine. Azurin contains a a carbonyl group of a glycine residue. T1Cu-containing

proteins are usually called cupredoxins. They work as electron transfer.

Type II copper centers (T2Cu, found in Nature) coordination by N or N/O ligands. T2Cu centers

occur in enzymes, where they assist in oxidations or oxygenations.

Type III copper centers (T3Cu, found in Nature) consist of a pair of copper centers, each

coordinated by three histidine residues. These centres are present in some oxidases and oxygen-

transporting proteins.

Diverse type of oxygen activating enzymes may use even the same substrate to perform same

reaction (see TH and Tyrosinase) but directed towards diverse metabolic path (melanin synthesis

vs adrenalin).

Activation of the substrate does not always require its direct binding to the metal sites to begin

catalyses (see PAM). To exert entire catalyses more than one functional domain may be present in

the protein, which may exert different function.

Spectroscopic Fingerprints That Reveal the

Nature of Copper Coordination Environments

UV/Vis

CD

Type I

Laccase BO

0.13 nm

Type I

Type II, III

cluster

Type IIImet

E0 Cu(2+)/Cu(+) ~ + 0.20-1.00 V

E0 Cu(2+)/Cu(+) ~ + 0.35-0.50 V

E0 Cu(2+)/Cu(+) ~ + 0.30-0.36 V

Type I

Type IIImet

Type II

Laccase, BO, Ceruloplasmine

Type II

Redox potential

Laccase

UV/Vis

Type III (peroxo)

E0 Cu(2+)/Cu(+) ~ + 0.4-0.5 V

Limulus polyphemus

Hemocyanine from Carcinus aestuarii (Crab)

Type III Cu

Model

RB3LYP/LACVP*

C2h symmetry

Zoppellaro et al.

πσ* to Cu(II) transition

πV* to Cu(II) transition

Electron

Paramagnetic

Resonance (X-band,

9.4-9.6 GHz, T = 77 K)

rR (resonance Raman)

Niels, Zoppellaro, et al. (hemocyanine from

Carcinus ae., Chem. Lett. 2011 asap)

Solomon E. I. Chem. Rev. 1996, 96, 2563

rR

The case of T0 Copper

X-band EPR

UV-Vis

Does the Protein Active-Site Structure Determines Protein Function ?

CATECHOL OX

Biomimetic Chemistry (231)

From ISI (time span 1900-2009) Topic=(Binclear/Trinuclear) AND Topic=(copper complexes)

From ISI (time span 1900-2010) Topic=(copper complexes)

Binuclear copper complexes: Entry 3828

Trinuclear copper complexes: Entry 1452

BIOCHEMISTRY & MOLECULAR BIOLOGY (226)

PHARMACOLOGY & PHARMACY (3)

CHEMISTRY Multisciplynary (925)

BIOPHYSICS (56)

SPECTROSCOPY (90)

BIOCHEMISTRY & MOLECULAR BIOLOGY (44)

CHEMISTRY, INORGANIC & NUCLEAR (22778)

CHEMISTRY, MULTIDISCIPLINARY (14581)

CRYSTALLOGRAPHY (6350)

CHEMISTRY, PHYSICAL (5842)

PHARMACOLOGY & PHARMACY (3)

CHEMISTRY Multisciplynary (303)

SPECTROSCOPY (19) BIOPHYSICS (4)

Crystallography (179)

Crystallography (495)

Biologically inspired oxidation catalysis

Lawrence Que, Jr & William B. Tolman

Nature 455, 333-340(18 September 2008)

Galactose oxidase

Biologically inspired oxidation catalysis

Lawrence Que, Jr & William B. Tolman

Nature 455, 333-340(18 September 2008)

Galactose oxidase Model

(Synthesis) Attempts to reproduce the coordination environment present in the active site of the target enzyme with e.g. self-assembly of discrete units into more complex structures or by following alternative approaches

Some mimiking complexes can replicate optical and/or magnetic properties of the enzymatic systems (e.g. type I and type III Cu’s)

Copper complexes designed as catecholase biomimics can activate O2 but oxidize only electron-rich catechols (e.g. DTBC)

None tested in water (very few in water/organic mixtures)

Stereoselective oxidation of hormonally active catecholamine little explored

(Electronics)

(Catalyses)

Case study:

Enzymatic systems

we further explore

Kitajma, JACS 1992, 1277

Karlin, JACS 1984, 2121

O2

Cu….Cu = 0.8940 nm

First Synthetic Model Complexes of CO/Ty/Hc

UV/Vis, Acetone, -80 0C

Cu….Cu = 0.3740 nm

Cu….Cu = 0.3240 nm

Cu….Cu = 0.3540 nm

Cu….Cu = 0.4550 nm

F. M

eyer

, C

hem

Eu

r. J

. 2

00

2 Cu….Cu = 0.2864 nm

Cu….Cu = 0.3447 nm

D. Das, Inorg. Chem. 2008, 7083

kcat = 3.24 x 104 h-1

KM = 0.0023 M

OH

OH

1/2 O2 O

O

H2O

….a

nd

Mo

re Ov

er the Y

ears (C

O/T

y/H

c mim

ics)

Ex

am

ple

of T

he

or

etic

al W

or

k o

n C

O

Sieg

ba

hn

Per E

. M. J

. Bio

l. Ino

rg. C

hem

. 20

07

, 125

1

Tyrosinase Reactivity in a Model Complex:

An Alternative Hydroxylation Mechanism

Liviu M. Mirica et al., Science 2005, 1890

UV-Vis spectrum of A (solid line) and PDBED (dashed line) in

MeTHF (153 K, [Cu] 1 mM). Inset: Resonance Raman spectra of

A ( ex = 413 nm, MeTHF, 77 K, [Cu] 1 mM) with 16O2/16OPhtBu2

(top line), 18O2/16OPhtBu2 (middle line), or 16O2/

18OPhtBu2 (bottom

line).

16O2

18O2

Chem. Eur. J. 2008, 3535

Inorg. Chem. 2003, 42, 5660

Trinuclear Cu Systems

Solomon E.I. Science 1996, 273, 1848

Wang, Inorg. Chem. (commun.) 2004, 7, 858

Solomon E.I. PNAS 2007, 13609

Zoppellaro G. J. Biol. Chem. 1999, 32718

B3LYP/TZVP

Example of Theoretical Work on Trinuclear Cu Cluster

NN

N

NN

AcOH2C

N

N

CH2OAc

N

N

N

N N

N

Ph

Ac

Site B

Site A Site A

(S)-Pz-(C2-(HisIm))2

Increasing Ligand

Complexity

Synthesis N

N

O+

N

N

N

NHHN

COOH

2 HCl(Boc)2O

H2O/dios.NN

COOH

BocBocNN BocBoc

O

NH

Ph

NH2H2N

O

NH

Ph

PhCH2NH2

HBTU/Et3N

DMF

NN

O

NH

Ph

CF3COOH

CH2Cl2

Cl

OO

Cl

NN

O

NH

Ph

N

OO

N

H3COOC

N

N

COOCH3

N

N

N

NN

NNN

O

NH

Ph

NH

OO

HN

H3COOC

N

N

COOCH3

N

N

NN

NH

Ph

NN

HOH2C

N

N

CH2OH

N

N

N

NN

NNN

N

NN

AcOH2C

N

N

CH2OAc

N

N

N

N N

N

Ph

2) NaHCO3 / CHCl3

1) NH3 (g) / CH2Cl2

NH2

H3COOC

NN

+ 2

Et3N

NMP

ClN

N

K2CO3

DMF

BH3 Me2S

THF

AcOAc

ClCl

O

+ 2

(1) (2)(3)

(4)(5)

(6) (7)

(8)(9)

Ac

Binuclear Copper System. Modeling

1.3 nm (<S*S>=2.0027)

DFT/RI-PB/def2-TZVP

MMFF94/Monte Carlo

(heating cycles 300-5000 K

Gradients 1 K/ps)

6400 conformations

E < 4.9 Kcal/mol

SO

MO

A1 –

SO

MO

A2 =

0.0

2 e

V

Trinuclear Copper System Modeling

(<S*S>=3.7559) DFT/RI-PB/def2-TZVP

MMFF94/Monte Carlo

(heating cycles 300-5000 K

Gradients 1 K/ps)

3844 conformations

E < 37.6 Kcal/mol

NN

N

NN

AcOH2C

NN

CH2OAc

N

N

N

NN

NPh

Ac

Cu2+Cu2+

NN

N

NN

AcOH2C

NN

CH2OAc

N

N

N

NN

NPh

Ac

Cu2+Cu2+

Cu2+

Electronic Fingerprints (UV/Vis and CD)

Free complex

+ N3-

+ N3-

+ N3-

Cu2L + 2N3-

Cu2L

Cu2-L Cu2L-(N3)2

gzz 2.238 2.231

gyy 2.066 2.079

gxx 2.066 2.038

Azz 146 × 10−4 cm−1 153 × 10−4 cm−1

Ayy 14 × 10−4 cm−1 10 × 10−4 cm−1

Axx 14 × 10−4 cm−1 9 × 10−4 cm−1

L/Ga 0.63 0.63

LWxb 84 × 10−4 cm−1 34 × 10−4 cm−1

LWyb 130 × 10−4 cm−1 102 × 10−4 cm−1

LWzb 88 × 10−4 cm−1 62 × 10−4 cm−1

STc 1.96 ± 0.12 1.85 ± 0.11

Bin

uclea

r Co

pp

er Sy

stem-E

PR

(T =

77

K)

(a)L/G indicates the Lorentzian/Gaussian line−shape.

(b)LWx,y,z indicates the line−width tensor. (c) ST indicates

the total Spin−concentration calculated against Cu(II)-EDTA

standard, 1.0 mM.

Obs Sim

Obs Sim

Trin

uclea

r Co

pp

er Sy

stem-E

PR

(T =

77 K

)

Cu3L + N3-

Cu3L

Cu3L + 3N3-

Cu3-L

gzz 2.233

gyy 2.092

gxx 2.081

Azz 145 × 10−4 cm−1

Ayy d n.d.

Axx d n.d.

L/Ga 1.00

LWxb 93 × 10−4 cm−1

LWyb 159 × 10−4 cm−1

LWzb 103 × 10−4 cm−1

STc 1.74 ± 0.11

Cu3L+(N3)3 Cu(1) Cu(2)

gzz 2.243 2.430

gyy 2.081 2.086

gxx 2.036 2.086

Azz 153 × 10−4 cm−1 102 × 10−4 cm−1

Ayy 18 × 10−4 cm−1 17 × 10−4 cm−1

Axx 10 × 10−4 cm−1 17 × 10−4 cm−1

L/Ga 0.70 0.67

LWxb 34 × 10−4 cm−1 32 × 10−4 cm−1

LWyb 103 × 10−4 cm−1 24 × 10−4 cm−1

LWzb 66 × 10−4 cm−1 30 × 10−4 cm−1

STc,f 2.35 ± 0.13

Cu3L Cu3LN3

Cu3L(N3)3

N3-

2N3-

1.3 K

Binuclear Cu

Trinuclear Cu

S = 3/2

S = 1/2

S = 1/2

S = 1/2

EPR, T = 3.6 K

Trinuclear Cu: spin-configurations

D ms = 1

D ms = 2

D ms = 2

16 mW

2 mW

From Theory to Experiments D ms = 2

Oxidation of L-DOPA to Chinone Use of freeze-quench EPR

Obs

Sim

ST ~ 1.0

ST < 0.1

Trinuclear Cu

T = 77 K

T = 77 K

Binuclear Cu 3-methyl-2-benzothiazolinone

hydrazone

SSkK

SVV

catM

)1(2

max

The dependence of the rates of the catalytic

reactions as a function of the substrate

concentration is biphasic. However all the

complexes feature substrate (S) inhibition at

high-substrate concentrations, and therefore

the kinetic parameters (Kcat, KM) were

estimated with the eq. 1

(2)

(1)

Under Turn-Over.

The Catalytic-Cycle

Oxidation of catechols and flavonoids:L-Dopa, D-Dopa, L-DopaOMe, D-DopaOMe, L-norepinephrine, D-norepinephrine, catechin and epichatechin (pH 7.0, PBS buffer/methanol, 20/1)

100)/()/(

)/()/(%

LMcatDMcat

LMcatDMcat

KkKk

KkKkR

The different degree of stereoselectivity

obtained for the binuclear and trinuclear

copper complexes were extracted through

the use of eq 2:

HO

HOOH

O

NH2

HO

OH

H2N

HO

[Cu2−(S)−Pz−(2C−His−Im)2][ClO4]4

Substrate KM [M]

kcat [s−1]

kcat/KM [M−1 s−1]

R % r2 [a]

(5.95±0.46)10−5 (1.42±0.03)10−2 239 33 0.99

(1.05±0.15)10−4 (1.28±0.05)10−2 122 0.97

(4.75±0.52)10−5 (2.58±0.06)10−

2 543 21 0.98

(4.90±0.32)10−5 (1.72±0.03)10−2 352 0.99

[Cu3−(S)−Pz−(2C−His−Im)2][ClO4]6

(3.84±0.41)10−5 (1.51±0.04)10−2 390 75 0.97

(2.48±0.27)10−4 (1.43±0.06)10−2 57 0.99

(2.60±0.37)10−4 (3.65±0.18)10−2 140 6 0.99

(3.43±0.42)10−4 (5.45±0.24)10−2 160 0.99

HO

HOOH

O

NH2

HO

OH

H2N

HO

HO

HOOH

O

NH2

HO

OH

H2N

HO

HO

HOOH

O

NH2

HO

OH

H2N

HO

L

D

L

D

L

D

L

D

Results

[a] The coefficient of determination (r2). In methanol/aqueous phosphate buffer, 50 mM, pH 7.0, with MBTH at 20 °C.

Conclusion

Optical/magnetic/structural properties can be

implemented into synthetic biomimic by design

Stereoselectivity versus a specific substrate (system

chemical property) remains difficult to implement into

a simple mimiking molecular architecture

….molecular complexity needs to be considered (and

included) within design