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F8390
Metalloproteins: Structure and Function
1. Introduction
1.1. Metalloproteins: Functions in Biological Chemistry
1.2. Some fundamental metal sites in metalloproteins
2. Mononuclear zinc enzymes: Carbonic anhydrase
3. Metalloproteins reacting with oxygen
3.1. Why do aerobic organisms need metalloproteins?
3.2. Oxygen transport proteins & Oxygenases
3.2.1. Hemoglobin, Myoglobin Cytochrome P450
3.2.2. Hemerythrin & Ribonucleotide Reductase R2 &
Methane monooxygenase diiron subunits
3.2.3. Hemocyanin & Tyrosinase
4. Electron transfer proteins
4.1. Iron-sulfur proteins
4.2. Blue copper proteins
5. Conclusion
3. Metalloproteins reacting with oxygen
3.1. Why do aerobic organisms need metalloproteins?
Cells of aerobic organisms need oxygen. First, oxygen is needed to gain energy
from food (respiration) and for other processes. Second, toxic organic substances
are eliminated from the body by oxidation, whereupon OH-groups are attached
to the molecule (this specific process is called hydroxylation, in mammals it
occurs mainly in the liver). This renders the toxic molecule water-soluble and it
can be eliminated (through the urine in mammals).
Cellular respiration
C6H12O6 + 6 O2  6 CO2 + 6 H2O
DG0 = -674 kcal/mol
Elimination of xenobiotics. Example: hydroxalation of hexane by Cytochrome P450
OH
n-Hexane
1-Hexanol
minor
OH
+ 1/2 O2
3-Hexanol
minor
2-Hexanol
major
Cytochrome P450
OH
Use of oxygen by aerobic organisms is hampered by two problems:
1. The solubility problem
Water solubility of oxygen at 25oC and pressure = 1 bar is at 40 mg/L water.
This is not enough to guarantee the oxygen supply to mitochondria by mere
diffusion. Cells of aerobic organisms use therefore oxygen transporters.
2. The kinetic problem
Oxygen has two unpaired electrons in its ground state and forms therefore a
triplet state. The overwhelming majority of organic molecules (such as glucose
or n-hexane) have all electrons paired and occur therefore in the singlet state.
The products of oxidation of organic molecules, CO2 and H2O, are also in
singlet states.
According to the so-called Wigner-rule, processes in which the spin-state
changes are « spin-forbidden », that is, they have a large kinetic barrier. The
solution of the problem is binding of O2 to a transition metal complex. In
transition metal complexes, spin-state changes are less inhibited due to the
spin-orbit coupling. The oxygen-bound metal complex can therefore transit
from a triplet state to a singlet state, and then react with an organic substrate
which has also a singlet ground-state.
Activation of O2 with the help of a transition metal complex:
Adduct formation from a pentacoordinated [FeL5]2+ complex and O2
_
_
O_
O_
_
_
O
L
L
2+
Fe
L
L
L
L
L
O_
2+
Fe
L
L
L
Molecular orbital level diagram for O2: 3Sg- state
2p
2p
2s
2s
O
O2
O
Bonding and antibonding MOs formed by
AOs 2p in the O2 molecule
antibonding
s*2p
p*2ph
xz
p*2pv
yz
p2ph
bonding
y
x
xz
p2pv
yz
z
s2p
Bonding and antibonding MOs forming a p
bond in the O2 molecule
antibonding
bonding
y
x
z
O
O
p*2pv
p*2ph
O
O
p2pv
p2ph
Activation of O2 with the help of a transition metal complex:
Adduct formation from a pentacoordinated [FeL5]2+ complex and O2
_
_
O_
O_
_
_
O
L
L
2+
Fe
L
L
L
L
L
O_
2+
Fe
L
L
L
Splitting of d orbitals in an octahedral environment
(6 equal ligands)
Cetral transition metal atom
Lone-pairs of ligands
z2 x2-y2
xy xz yz
L
L
L
M
M
L
L
L
Splitting of d orbitals in an tetragonal environment
(5 equal ligands)
Cetral transition metal atom
Lone-pairs of ligands
6 ligands
octahedral
field
5 ligands
octahedral
field
z2 x2-y2
xy xz yz
L
L
L
L
L
L
L
M
M
L
L
M
L
L
Splitting of d orbitals in an tetragonal environment
(5 equal ligands)
Cetral transition metal atom
Lone-pairs of ligands
6 ligands
octahedral
field
5 ligands
octahedral
field
z2 x2-y2
x2-y2
z2
xy xz yz
L
L
L
L
L
L
L
M
M
L
L
M
L
L
Splitting of d orbitals in an tetragonal environment
(5 equal ligands)
Cetral transition metal atom
FeII is a d6 ion
Remember for next slide
Lone-pairs of ligands
6 ligands
octahedral
field
5 ligands
octahedral
field
z2 x2-y2
x2-y2
z2
xy xz yz
xy
xz
xz
L
L
L
L
L
L
L
M
M
L
L
M
L
L
+ 1[L5Fe] 
3[L FeO ]
spin-allowed:
5
2
n° of unpaired electrons unchanged
3O
From last slide
2
3O
2
s*
One of the p* orbitals
of O2 overlaps with
the dz2 orbital of Fe
and forms a bond; the
other p* orbital is
non-bonding
dx2-y2
dz 2
dz2 empty
L
p*
dxy
dxz,dyz
O2
(only the two unpaired
valence electrons shown)
3
[L5FeO2]
L
L
1[L Fe]
5
s
3
L
Fe2+
1
[L5Fe]
L
+ 1[L5Fe] 
3[L FeO ]
spin-allowed:
5
2
n° of unpaired electrons unchanged
3O
2
3O
2
s*
One of the p* orbitals
of O2 overlaps with
the dz2 orbital of Fe
and forms a bond; the
other p* orbital is
non-bonding
dx2-y2
d z2
L
p*
L
dxy
dxz,dyz
(only the two unpaired
valence electrons shown)
3
[L5FeO2]
1
[L5Fe]
L
13[L
1[LFeO
5 5Fe]2]
s
O2
Fe2+
L
Stabilization!
3
L
1
[L5FeO2]
process spin-forbidden
but rendered possible
by spin-orbit coupling
In transition metal complexes, spin-orbit coupling renders
spin-forbidden transitions possible.
Metal complexes can therefore activate (triplet) oxygen for reactions
with (singlet) organic molecules.
[MLn]m+ + 3O2
1[ML
m+
O
]
n 2
+ 1[Substrate]
1[Oxidation
Metal-oxygen adducts can also be used
as oxygen carriers!
2. Oxygen transport proteins & oxygenases
products]
Oxygen transport proteins: O2 binding in active sites
Hemoglobin
(vertebrates, some invertebrates)
Hemocyanin
(molluscs, some arthropods)
Hemerythrin
(some marine invertebrates)
Lippard: Bioinorganic Chemistry, 1994
2.2.1. Hemoglobin & Myoglobin
in vertebrates
2
2e-
Respiration:
Reduction of O2 to H2O
by cytochrome c
catalyzed by the enzyme
cytochrome-oxidase
153 amino acids
http://www.ul.ie/~childsp/CinA/Issue64/TOC36_Haemoglobin.htm
Mechanism of O2 binding to Fe in myoglobin and hemoglobin
_
_
O_
_
O_
O
O_
_
O
N
x
Fe
N
N
N
Fe
+
N
_
N
N
N
+
Orbital energy difference is
Nowthan
let us
make the
smaller
spin-pairing
energy
difference
energy.
Unpaired
electrons are
between dxz,yz and coupled
p*n
antiferromagnetically
larger
 diamagnetic
state
_
y
+
N
Fe
N
+
N
N
N
N
+
N
O_
_
_
+
N
H
_
N
s*
N
H
H
x2-y2
x2-y2
z2
x2-y2
Weiss
Nature 1964, 202,
83-84; 203, 183
z2
p*n
xy
xz,yz
xy
xz,yz
xy
p*
xz,yz
s
High-spin Fe(II)
Low-spin Fe(II)
Low-spin Fe(III)-O2-
O2
_
_
O_
_
O_
O
O_
_
O
N
x
Fe
N
N
N
Fe
+
N
_
N
N
N
+
Orbital energy difference
is larger than spin-pairing
energy  electron will
go to dyz
_
y
+
N
Fe
N
+
N
N
N
N
+
N
O_
_
_
+
N
H
_
N
s*
N
H
H
z
x2-y2
x2-y2
z2
x2-y2
xy
Nature 1964
203, 182-183
p*n
2
xz,yz
Pauling
xy
xz,yz
xy
p*
xz,yz
s
High-spin Fe(II)
Low-spin Fe(II)
Low-spin Fe(II)-O2
O2
Fe(II)-O2, Fe(III)-O2-, or Fe(IV)-O22-?
FeIV
_ _
O
Fe
_
_ _
_
_O
Fe(IV)-O22-
.
FeIII
.
Pauling
dioxygen
.
_ _
FeIII
_
_
O
_O
_
_O
_
_O
Fe(II)-O20
peroxide
_
O
_
O
II
Fe(III)-O2-
_
.
Weiss
superoxide
What experimental data can be used to determine whether oxygen in
oxyhemoglobin resembles more to Fe(III)-O2- or to Fe(II)-O2?
Stretching frequencies and bond lengths in dioxygen species
Species
[A]
nO-O [cm-1]
d O-O
O2 +
1905
1.12
O2
1580
1.21
O2 -
1097
1.33
O22-
802
1.49
M-O2-
1100-1150
1.24-1.31
M- O22-
800-900
1.35-1.50
1105
1.22
Mb-O2
Oxymyoglobin resembles FeIII-O2-
O2 versus CO discrimination
Isolated heme has a 104 times larger affinity for CO than for O2.
In myoglobin & hemoglobin, this discrimination factor drops to 102. Why?
H
_N
H
N
N
_
O
_O
N
_
N
H
_
+
+
Fe
III
+
+
N
N
_
N
Quantum chemical calculations indicate that the terminal O-atom
is more negatively charged in the O2 complex than in the CO-complex.
H
_N
H
+
+
N
N
_
N
N
+
+
Fe
N
III
C
N
_
_O
N
H
Hydrogen bonding with distal histidine favors O2-binding
against CO-binding .
Hemoglobin/Myoglobin: History
1936
L.Pauling: Oxyhemoglobin is diamagnetic
1938-
M. F. Perutz & J. C. Kendrew: x-ray studies on Hb & Mb
1939
L. Pauling: measurements of pKa  2 histidines,
1 coordinated + 1 farther from Fe
distal
proximal
histidine
pKa=6.8
histidine
pKa=5.7
1960
J. C. Kendrew: Crystal structure of myoglobin at 2 A
1964
J. J. Weiss: Oxyhemoglobin is a Fe(III)-(O2-) complex
with antiferromagnetic coupling
1964
L.Pauling: Fe(II)-O2 complex, postulate of H-bond
1981
S. E. V. Phillips, B. P. Schoenhorn: H-bond confirmed by
neutron diffraction
Practical training
- Download from the pdb database the structure of human hemoglobin 1HHO
http://www.rcsb.org/pdb/home/home.do
The coordinate file contains one half of the tetrameric structure, with one
alpha and one beta subunit.
- Display the structure using VMD
- Highlight (using Graphics/Representation,
Selected atoms „name FE“, drawing method
CPK) the iron atoms of each subunit
- Identify the residue numbers of both heme
residues and highlight them
- Identify the proximal and distal histidines
- Measure the distances O-NE2 and Fe-NE2
distances for both histidines, for both hemes
- Carry out the same for the CO-Hemoglobin
complex 1HCO
- Interpret your observations