2 Hemocyanin

Chapter I.
1:
Hemocyanin (Hc)
lundi 11 juillet 2011
O2 is essential for animals

Low O2 solubility in aqueous solution (0.2 ml in 100 ml plasma)
need some ways to carry O2
Exception
Notothenoids/family Channichthyidae
O2 absorbed and distributed directly by
the plasma
Can survive without O2-carrier because
of:
the high O2 content of the cold waters
of the Southern Ocean
u
the good solubility of O2 due to the

high pressure
u
Antarctic Icefish
(body temperature: -1.7oC)
Dioxygen reduction
e-
O2
e -, 2 H +
O2-
Thermodinamically unfavorable
e -, H +
e -, H +
H2O2
H2O
HO
H2O
O2-
negative reduction potentiel

E = - 0.33 V
Kinetically unfavorable
O2
spin state restriction

In ground state O2 is in triplet state (3O2)
with 2 unpaired electrons in 2p* orbital.
Most of organic substances have a singlet
ground states (no unpaired electron).
unfavorable reactions
H2O2
E = + 0.82 V
HO
H2O
Respiratory metal-containing
proteins
Hemoglobin (Hb): Fe
vertebrates (blood cells), echinoderms, annelids,
molluscs, insect larvae
Chlorocurorin: Fe (similar to myoglobin)

annelids
Hemerythrin (Hr): Fe
marine invertebrates, annelids
Hemocyanin (Hc): Cu
molluscs and arthropods
Dioxygen Carriers
Properties
Hemoglobin Hemerythrin Hemocyanin
Ox. state in deoxy form
(II)
(II)
(I)
Metaln:O2
Fe:O2
Fe2:O2
Cu2:O2
Color deoxy form
Red-purple
Colorless
Colorless
Color oxy form
Red
Violet-pink
Blue
Metal coordination
Porphyrin ring
Prot. side chain

(Glu, His)
Prot. side chain

(His)
mol. weight (kD)
65
108
400 to 20 000
numb. subunits
many
Hemoglobin (Hb)
Hemoglobin is an 22 heterodimer
2
1
-chain
oxy-Hemoglobin
O
FeIII
Back
Hemerythrin (Hr)
NHis
NHis
O
FeII
NHis
H O
O
O
NHis
FeII
O
O
O2
NHis
NHis
FeIII
FeIII
NHis
NHis
O
O
O
O
O2
deoxy-Hr
NHis
NHis
Back
oxy-Hr
Hemocyanin (Hc)
H
N
H
N
HN
N
(I)
Cu
N
HN
H
N
(I)
O2
N
Cu
NH
HN
N
N
HN
H
N
(II)
Cu (O2) Cu
N
HN
(II)
N
HN
NH
Hemocyanin (Hc)
Hc are respiratory proteins containing 2 Cuatoms that reversibly bind a single O2.
Oxygenation causes a color change between
the colorless Cu(I) deoxygenated form and
the blue Cu(II) oxygenated form.
Hc carry O2 in the blood of most molluscs,
and some arthropods. They are second only
to Hb in biological popularity of use in O2
transport.
Hemocyanin (Hc).
Hc is made of individual subunit proteins which contains
1 active site.
Each subunit weighs about 75 kDa. Subunits are arranged
in chains or bundles in weights exceeding 1500 kDa.
Because of the large size of Hc, it is found free-floating in
the blood, unlike Hb, which must be contained in cells
because its small size would lead it to clog and damage
blood filtering organs such as the kidneys. This freefloating nature allows for higher densities of Hc in the
blood (as compared to Hb), and helps offset its low
efficiency.
Crystal structure of hexameric Hc

Panulirus interruptus (3.2 resolution)
x6
Volbeda, A., Hol, W. G. et al. (1989) J. Mol. Biol. 209: 249-279

90
Oligomeric forms of Hc
6 sub-u
12 sub-u
x2
24 sub-u
x2
Decker H. et al. (2009) Structure 17(5): 749-758

Hc active site
dCu-Cu = 3.8
Oxy form of Hemocyanin (oxy-Hc)

H
N
H
N
H
N
HN
N
(I)
Cu
N
HN
(I)
N
Cu
NH
O2
HN
N
N
HN
H
N
(II)
(II)
Cu (O2) Cu
NH
N
HN
HN
EXAFS: dCu-Cu = 3.67

X-ray: dCu-Cu = 3.8
EPR: Silent
Raman: (O-O) = 749 cm-1

UV-vis.: 345 nm ( = 20 000 M-1 cm-1)
550 nm ( = 1000 M-1 cm-1)
EPR: Silent
Coordination modes for O2 to

a binuclear Cu(II) center
2-
H
O
Cu
O
Cu
O
Terminal
-1 (end-on)
H
O
Cu
O
Cu
O
Cu
Cu
O
cis--1,2
trans--1,2
-1:1 (end-on)
O
Cu
Cu
-1,1
From enzyme to model and vice versa

Metalloprotein
RH + O2
ROH + H2O
Cu
Cu
Cu
Cu
Biomimetic/bioinspired
model
RH + O2 ROH + H2O
Biomimetic vs. bioinspired models

Ornithoptere (XVIe century)
AirBus A380 (XXIe century)
meta-Xylyl ligand
Br
OH Br
N
H
OH N
N
N
K. D. Karlin et al. J. Am. Chem. Soc. 1984, 106, 3372.

Terminal peroxo
O2
N
O
Cu
Cu
Raman: (O-O) = 749 cm-1

UV-vis.: 345 nm ( = 20 000
M-1 cm-1)
550 nm ( = 1000 M-1 cm-1)
CH2Cl2
184 K
Cu
k-1
EPR: Silent
k1
Cu
O O
k1 > 106 M-1s-1

UV-vis.: 385 nm ( = 20 000 M-1 cm-1)
505 nm ( = 3000 M-1 cm-1)
EPR: Silent
K. D. Karlin et al. J. Am. Chem. Soc. 1984, 106, 3372;

1987, 109, 2668; 1988, 110, 6882.
O
Cu
O2
N
Cu I
CH2Cl2
r. t.
II
II
Cu
Cu
N
N
N
1 e- reduction
O
Cu
N
I
O2
N
II
Cu
N
CH2Cl2
r. t.
II
II
Cu
Cu
N
N
N
K. D. Karlin et al. J. Am. Chem. Soc. 1992, 114, 7599;

Inorg. Chem. 1992, 31, 3001.
tris-(2-pyridylmethyl)amine (TPA)
N
(iPr)2EtNH
NH2
+
N
Br
N
NH2
R1
+
O
R2
NaBH4
R1
N
R1
N
NH
R2 (iPr)2EtNH
N
R3
R2
Br
R3
1
1
- :
peroxo (end-on)
O2
N
N
k1
N
Cu
N
N
N
N
Cu
O
k-1
EtCN or THF
183 K
N
Cu

Raman: (O-O) = 749 cm-1
X-ray: dCu-Cu = 4.36
UV-vis.: 345 nm ( = 20 000
UV-vis.: 525 nm ( = 11 500 M-1 cm-1)
M-1 cm-1)
590 nm ( = 7600 M-1 cm-1)
550 nm ( = 1000 M-1 cm-1)
EPR: Silent
EPR: Silent
Substituted TPA
R
R
R
N
N
N
Cu
O2
k1
N
k-1
k1 = 1.34/1.85 104 M-1s-1

k-1 = 2/0.8 10-5 M-1s-1
K = 0.67/24 108
Cu
N
EtCN
183 K
R = H & MeO
N
O
N
Cu
N
R
H = -47.2/-49 kJ mol-1
S = -89/-87 J K-1 mol-1
Pyrazolylborates (scorpionates)
R
NaBH4
N NH
N
H
N
N N
B
N
anisole
R
- H2
R
K
2
2
- :
R
H
R
N N
Cu
NN
O2
k-1
EtCN or THF
183 K
k1
S
R
peroxo
H
R
N N
Cu
NN
R
O
O
R
R
N N
Cu
NN N
X-ray: dCu-Cu = 3.56
Raman: (O-O) = 749 cm-1
Raman: (O-O) = 741 cm-1
UV-vis.: 345 nm ( = 20 000 M-1 cm-1)
UV-vis.: 349 nm ( = 21 000 M-1 cm-1)
550 nm ( = 1000 M-1 cm-1)
551 nm ( = 800 M-1 cm-1)
EPR: Silent
EPR: Silent
Kitajima et al. J. Am. Chem. Soc. 1989, 111, 8975.

Structure of oxy-Hc
limulus polyphemus
K. A. Magnus et al. Chem. Rev. 1994, 94, 727.

O2
k1
N
N
Cu
Cu
N
k-1
CH2Cl2
184 K
Cu
Cu
O
N
N
unstable
R
H
tBu
F
NO2
k1 x 102(M-1s-1)
3.85
4.90
0.093
1.09
k-1 x 10-5 (M-1s-1)

1.5
0.5
0.3
1.8
K x 107
2.6
9.6
0.33
0.59
The life time of the dioxygen adduct is increased when R is

electron withdrawing group enabling characterization by
resonance Raman spectroscopy (only for R = NO2).
Biomimetic vs. bioinspired Hc models

O2
N
N
Cu
CH2Cl2
184 K
Cu
N
Cu
Cu
O
k1 = 3.85 102 M-1s-1
Biomimetic
O2
N
O
Cu
Cu
N
CH2Cl2
184 K
k1 > 106 M-1s-1

Cu
N
Cu
O O
Bioinspired
Spacer
N
Spacer
N
Cu
O2
N
II
II
Cu
Cu
N
Cu
O2
()n N
Cu
k1 = 8.5 103 M-1s-1

K = 7 107
Cu
N
O2
O Cu
Cu
O
Cu
O
H = -58 kJ mol-1
S = -165 J K-1 mol-1
k1 = 11 103 M-1s-1
K = 4.5 102
CH2Cl2
184 K
Cu
N
O2
N
N
H = -28 kJ mol-1
S = -101 J K-1 mol-1
RT stable Cu2:O2 adduct
Kodera M. et al. (1999) J Am Chem Soc 121(47):1100611007

O2
Spacer
LCu(I)
Cu(I)L
k1
Spacer
LCu(II)
k-1
O2
LCu(II)
Cu(II)L
Spacer
LCu(II)
k-1
LCu(II)
O2
Cu(II)L
O2
Spacer
Cu(II)L
O2
Cu(I)L
O2
Spacer
LCu(II)
k1
Spacer
Spacer
Cu(II)L
LCu(II)
O2
O2
LCu(II)
Cu(II)L
Spacer
Cu(II)L
O2
1,4,7-Triazacyclononane (TACN)
Ts
Ts
N
N
H
+
TsO
1) CsCO3/DMF
2) H2SO4
3) RX/NEt3
O2
N
k1
Cu
N
R
k-1
EtCN or THF
183 K
R
O
Cu
Cu
N
R
N
N
W. B. Tolman et al. Science 1996, 271, 1397.

N
R
OTs
N
R
N
H
Ts
Ph
()m
N
II I
()n
Cu
CT CuIIO22O2
Absorbance
1.5
Ph
()m
d-d
0.5
N
Cu
0
389
459
529
599
669
()n
Cu
(nm)
()n
N
()m
Ph
n or m = 0 or 1
Eugnie Peyroux, Marseille Thesis 2008, December

LCu(I)
O2
k1
LCu(I)
LCu(II)O2
fast
k-1
LCu(II)O2Cu(II)L
rate determining step
CH3
N
N
II I
Cu
k1 = 1.91 104 M-1s-1

k-1 = 3.23 10-2 M-1s-1
K = 5.9 105
H = -0.7 kJ mol-1
S = -164 J K-1 mol-1
H = -89 kJ mol-1
S = -240 J K-1 mol-1

2 Hemocyanin

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Chapter I.

lundi 11 juillet 2011

O2 is essential for animals

the good solubility of O2 due to the

lundi 11 juillet 2011

negative reduction potentiel

spin state restriction

Chlorocurorin: Fe (similar to myoglobin)

Hemoglobin Hemerythrin Hemocyanin

Ox. state in deoxy form

Color deoxy form

Color oxy form

Prot. side chain

Prot. side chain

mol. weight (kD)

lundi 11 juillet 2011

lundi 11 juillet 2011

lundi 11 juillet 2011

Crystal structure of hexameric Hc

Volbeda, A., Hol, W. G. et al. (1989) J. Mol. Biol. 209: 249-279

Volbeda, A., Hol, W. G. et al. (1989) J. Mol. Biol. 209: 249-279

lundi 11 juillet 2011

Decker H. et al. (2009) Structure 17(5): 749-758

Oxy form of Hemocyanin (oxy-Hc)

EXAFS: dCu-Cu = 3.67

Raman: (O-O) = 749 cm-1

lundi 11 juillet 2011

Coordination modes for O2 to

lundi 11 juillet 2011

From enzyme to model and vice versa

Biomimetic vs. bioinspired models

AirBus A380 (XXIe century)

lundi 11 juillet 2011

K. D. Karlin et al. J. Am. Chem. Soc. 1984, 106, 3372.

Raman: (O-O) = 749 cm-1

EXAFS: dCu-Cu = 3.67

k1 > 106 M-1s-1

K. D. Karlin et al. J. Am. Chem. Soc. 1984, 106, 3372;

K. D. Karlin et al. J. Am. Chem. Soc. 1992, 114, 7599;

lundi 11 juillet 2011

EXAFS: dCu-Cu = 3.67

X-ray: dCu-Cu = 4.36

UV-vis.: 345 nm ( = 20 000

UV-vis.: 525 nm ( = 11 500 M-1 cm-1)

590 nm ( = 7600 M-1 cm-1)

550 nm ( = 1000 M-1 cm-1)

k1 = 1.34/1.85 104 M-1s-1

lundi 11 juillet 2011

EXAFS: dCu-Cu = 3.67

X-ray: dCu-Cu = 3.56

Raman: (O-O) = 749 cm-1

Raman: (O-O) = 741 cm-1

UV-vis.: 345 nm ( = 20 000 M-1 cm-1)

UV-vis.: 349 nm ( = 21 000 M-1 cm-1)

550 nm ( = 1000 M-1 cm-1)

551 nm ( = 800 M-1 cm-1)

Kitajima et al. J. Am. Chem. Soc. 1989, 111, 8975.

K. A. Magnus et al. Chem. Rev. 1994, 94, 727.

k-1 x 10-5 (M-1s-1)

The life time of the dioxygen adduct is increased when R is

Biomimetic vs. bioinspired Hc models

k1 = 3.85 102 M-1s-1

k1 > 106 M-1s-1