Transcript Metaloproteiny

F5351
Metalloproteins reacting with oxygen
1. Why do aerobic organisms need metalloproteins?
2. Oxygen transport proteins & Oxygenases
2.1. Hemoglobin, Myoglobin & Cytochrome P450
2.2. Hemerythrin & Methane monooxygenase
2.3. Hemocyanin & Tyrosinase
3. Conclusion
Jiří Kozelka
13.11. 2014
[email protected]
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.
Molecular orbital level diagram for O2: 3Sg- state
2p
2p
2s
2s
O
O2
O
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
Vazebné a antivazebné molekulové orbitály
tvořené atomovými orbitály 2p v molekule O2
antivazebné
s*2p
p*2ph
xz
p*2pv
yz
p2ph
vazebné
y
x
xz
p2pv
yz
z
Index h = horizontální
Index v´= vertikální
s2p
Vazebné a antivazebné molekulové orbitály tvořící vazbu p v molekule O2:
prostorové uspořádání
antivazebné
vazebné
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
6 ligands
octahedral
field
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
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
2
dx2-y2
dz 2
L
p*
dxy
dxz,dyz
3
O2
(only the two unpaired
valence electrons shown)
3
[L5FeO2]
1
[L5Fe]
L
Fe2+
L
L
L
+ 1[L5Fe] 
3[L FeO ]
spin-allowed:
5
2
n° of unpaired electrons unchanged
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
L
p*
dxy
dxz,dyz
s
3
O2
(only the two unpaired
valence electrons shown)
3
[L5FeO2]
1
[L5Fe]
L
Fe2+
L
L
L
+ 1[L5Fe] 
3[L FeO ]
spin-allowed:
5
2
n° of unpaired electrons unchanged
3O
2
_
_
O
s*
L
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
L
dx2-y2
O_
Fe2+
L
L
L
d z2
p*
dxy
dxz,dyz
spin inversion
s
3
O2
(only the two unpaired
valence electrons shown)
3
[L5FeO2]
1
[L5Fe]
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
_
_
O_
O2 oxygen molecule
O_
-_
_
O_
-
O2 superoxide anion
N
N
Fe
2+
N
N
N
N
N
Fe
.
O_
3+
N
N
N
Aminokyselina histidin tvořící koordinativní vazbu k Fe
„proximální histidin“. Toto je jediná kovalentní vazba mezi
porfyrinem železa a proteinem. Ostatní síly jsou hydrofobní,
mezi porfyrinovým cyklem a hydrofobními postranními
řetězci proteinu.
in vertebrates
2
2e-
Reduction of O2 to H2O
Catalyzed by the enzyme
Cytochrome-oxidase
2.1. Hemoglobin, Myoglobin & Cytochrome P450
153 amino acids
http://www.ul.ie/~childsp/CinA/Issue64/TOC36_Haemoglobin.htm
Vazba myoglobinu (Mb) na kyslík
Mb + O2
MbO2
Cvičení 1: definujte rovnovážnou konstantu pro zpětnou reakci
(tzv. disociační konstantu, Kd)
Cvičení 2: definujte saturaci vazebných míst, Y, definovanou rovnicí dole,
pomocí Kd a [O2] jako proměnných.
Nahraďte ve vzorcích pro Kd a pro Y koncentraci [O2] parciálním tlakem
p(O2).
Cvičení 3
Vypočtěte křivku frakční saturace kyslíku na myoglobinu. Disociační konstanta
komplexu MbO2 je, při 37 °C, pH = 7 a p = 760 Torr, Kd = 2.8 Torr.
p(O2) [Torr]
0.5
1
2
3
5
10
20
30
40
50
60
70
80
90
Y [%]
Y [%]
100
80
60
Cvičení 4: Jaký význam má
směrnice saturační křivky v bodě
p(O2) = 0?
Znázorněte graficky závislost
dY/dp(O2) na p(O2)
40
20
0
0
30
60
p(O2) [Torr]
90
Saturační křivka hemoglobinu
neodpovídá jedné jediné
rovnovážné reakci
Vazba O2 na jednu hemovou
skupinu hemoglobinu zvyšuje
afinitu pro O2 dalších jednotek
„Kooperativní efekt“
Cooperativity of oxygen binding by the 4 subunits of hemoglobin:
In deoxygenated form, the 4 subunits stabilize mutually the domed conformation.
The oxygen affinity of unloaded hemoglobin is smaller than that of individual
subunits. Oxygen binding to one subunit of hemoglobin favors the planar form
at neighboring subunits  fully loaded hemoglobin has an affinity similar to that
of an individual subunit.
http://www.chemistry.wustl.edu/~edudev/LabTutorials/Hemoglobin/MetalComplexinBlood.html
Effect of CO2 on oxygen afinity of hemoglobin: „Bohr-Effect“
In muscles, where metabolic activity produces CO2, amino groups
of certains amino acids are transformed to carbamate:
NH2
+
O
C
O
amino acid
amino acid
HN
N
+
H+
+
H+
O
The liberated H+ protonates histidine residues:
HN
O-
NH
N+
H
At subunit interfaces salt bridges are formed:
O
HN
N+
amino acid
H
O-
NH
These salt bridges favor the domed conformation  favor O2 release
 CO2 favors release of O2 which is then taken up by myoglobin
In muscles:
High CO2 concentration favors domed
conformation  favors O2 release
In bronchi:
Low CO2 concentration favors planar
conformation  favors O2 binding
http://www.chemistry.wustl.edu/~edudev/LabTutorials/Hemoglobin/MetalComplexinBlood.html
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-
F5351
Metalloproteins reacting with oxygen
1. Why do aerobic organisms need metalloproteins?
2. Oxygen transport proteins & Oxygenases
2.1. Hemoglobin, Myoglobin & Cytochrome P450
2.2. Hemerythrin & Methane monooxygenase
2.3. Hemocyanin & Tyrosinase
3. Conclusion
Jiří Kozelka
13.11. 2014
[email protected]
Hemoproteins: Axial Ligands and Functions
From: Cécile Claude, „Enzyme Models of Chloroperoxidase and Catalase“, Inaugural Dissertation, Universität Basel, 2001
Modification of the FeII/FeIII redox potential by the protein environment
Hemoprotein
proximal ligand
FeII (Red.) stable
Strong oxidants
Em for FeII/FeIII (mV)
FeIII/FeII (aq.)
FeIII/FeII
-
+770
Human hemoglobin
FeIII/FeII
His
+150
Microperoxidase11-CO
FeIII/FeII
His
+100
Chloroperoxidase
FeIII/FeII
Cys-
-150
NO synthase neuronal
FeIII/FeII
Cys-
-250
Horse-radish peroxidase
FeIII/FeII
His
-280
Cytochrome P450 2C5
FeIII/FeII
Cys-
-330
Catalase
FeIII/FeII
Tyr-
-460
FeIII (Ox.) stable
Strong reductants
Source: C. Capeillere-Blandin, D. Matthieu & D. Mansuy,
Biochem. J. 2005, 392, 583-587
Different metalloproteins need different redox potential for their function. Cytochrome
P450 needs to access the unusual oxidation state Fe(V) to be able to oxidize even
unreactive substrates. Therefore, it uses the negatively charged cysteine ligand which
donates electrons to Fe and stabilizes the high oxidation state. One of strategies that
proteins employ to modify the redox potential is using different proximal ligands.
Examples of Cytochrome P450 substrates
Hydroxylation at:
-aliphatic carbons
-aromatic carbons
-double bonds
-heteroatoms
local anesthetic
steroid hormone
carcinogen from fungi
antibiotic
Alkaloid from Taxus brevifolia, potent anti-cancer drug
Cytochrome P450cam (Campher-5-monooxygenase; pdb-code 1T86)
access for
substrate and O2
Hlavní dva rozdíly mezi hemoproteiny myoglobin a cytochrom P450,
důležité pro jejich různé funkce:
1. Přístupový kanál vedoucí ke kofaktoru (hemu) je u myoglobinu velmi
úzký, nedovoluje přístup větším molekulám než O2. U cytochromu P450
je kanál širší a v blízkosti kofaktoru obsahuje místo s vysokou afinitou
pro specifické substráty.
2. Distální cystein a okolí kofaktoru snižuje u cytochromu P450 oxidačněredukční potenciál Fe, takže tento metaloprotein může fungovat jako
oxygenáza a Fe v katalytickém cyklu může krátkodobě existovat v
oxidačním stupni Fe(V). Tento velmi reaktivní přechodný stav je
schopen hydroxylovat i poměrně nereaktivní alifatické atomy uhlíku.
F5351
Metalloproteins reacting with oxygen
1. Why do aerobic organisms need metalloproteins?
2. Oxygen transport proteins & Oxygenases
2.1. Hemoglobin, Myoglobin & Cytochrome P450
2.2. Hemerythrin & Methane monooxygenase
2.3. Hemocyanin & Tyrosinase
3. Conclusion
Jiří Kozelka
13.11. 2014
[email protected]
http://notes.chem.usyd.edu.au/course/codd/CHEM3105/Metalloproteins3.pdf
(HOI2)-
Crystal structure of hemerytrhin in unloaded state (pdb-code 1HMD)
Hexacoordinate Fe(II)
Pentacoordinate
Fe(II)
can bind O2
Dinuclear iron active site fixed by a four-helix bundle
Amino acids/subunit
153
113
628
Sipuncula
Priapulida
Brachiopoda
Hemerythrin je metaloprotein
transportující kyslík u některých
bezobratlých
Magelona papillicornis
Active sites of the reduced forms of Hemerythrin, Ribonucleotide Reductase R2 protein,
the hydroxylase component of Methane Monooxygenase, and D9 desaturase
Bridging carboxylates
Extra carboxylates stabilize higher oxidation states
Catalytic Cycle of soluble Methane Monooxygenase (sMMO)
This is a very strong
oxydant. The
carboxylate ligands
(preceding slide)
serve to stabilize the
high oxidation state
Fe(IV) of the two iron
atoms.
Methane is a very unreactive
compound. Needs an
extremely strong oxydant to be
hydroxylated.
Kopp & Lippard, Current Op. Chem. Biol. 2002, 568
F5351
Metalloproteins reacting with oxygen
1. Why do aerobic organisms need metalloproteins?
2. Oxygen transport proteins & Oxygenases
2.1. Hemoglobin, Myoglobin & Cytochrome P450
2.2. Hemerythrin & Methane monooxygenase
2.3. Hemocyanin & Tyrosinase
3. Conclusion
Jiří Kozelka
13.11. 2014
[email protected]
Amino acids/subunit
153
113
628
Hemocyanin je metaloprotein
transportující kyslík u většiny
měkkýšů a u některých korýšů
Panulirus interruptus
Linulus polyphemus
Octopus dofleini
Megathura crenulata
Hemocyanin: History
1878
Leon Federicq: Sur l‘hemocyanine, substance nouvelle
de sang de Poulpe (Octopus vulgaris)
(Compt. Rend. Acad. Sci. 87, 996-998)
Discovery
1901
M. Henze: Zur Kenntniss des Haemocyanins
Z. Physiol. Chem. 33, 370
Hemocyanin contains copper
1940
W. A. Rawlinson, Australian J. Exp. Biol. Med. Sci. 18,
131
Oxy-hemocyanin is diamagnetic
Známé a hypotetické (*) komplexy mědi s jednotkou O2
http://webdoc.sub.gwdg.de/diss/2003/ackermann/ackermann.pdf
On the search for functional hemocyanin model compounds
Karlin et al., JACS 1988, 110, 3690’3692
The first model complex showing reversible O2 binding by a dicopper unit
However, this complex differs from oxy-Hc:
Cu-Cu[Å]
1
1
υ(O-O)[cm-1]
4.36
Oxy-Hc 3.5-3.7
834
744-752
Karlin et al., J. Am. Chem. Soc. 1988, 110, 3690-3692
UV-VIS
440(2000)
525(11500)
590(7600)
1035(160)
340(20000)
580(100)
Model complex showing reversible O2 binding and similar features to Hc
Kitajima et al., J. Am. Chem. Soc.
1989, 111, 8975-8976
Cu-Cu[Å]
2
3.56
υ(O-O)[cm-1]
741
UV-VIS
349(21000)
551(790)
2
Oxy-Hc
3.5-3.7
744-752
340(20000)
580(100)
Functional hemocyanin models
[(tmpa)2Cu2O2]2+
[Cu{HB(3,5-iPr2pz)3}]2(O2)
Karlin et al., JACS 1988, 110, 3690’3692
Kitajima et al., JACS 1989, 111, 8975-8976
UV-Vis absorption spectra of the oxy forms of hemocyanin and tyrosinase
ps→d
pv→d
d→d
5-9 years later (1994, 1998):
Active sites in hemocyanins determined by X-ray crystallography
Magnus et al.,Proteins Struct. Funct. Gen.1994
Limulus polyphemus
Cuff et al.,J.Mol.Biol.1998
Octopus dofleini
An earlier model for hemocyanin...
…turned out to be a model for the enzyme tyrosinase!
Karlin et al., JACS 1984, 106, 2121-2128
L-DOPAquinone
Syntéza melaninu z tyrosinu katalyzovaná enzymem tyrosináza
http://pollux.chem.umn.edu/~kinsinge/new_homepage/research/gss_presentation_3/sld019.htm
Tyrosinase versus Hemocyanin
The coupled binuclear copper sites in tyrosinase and hemocyanin are very similar.
Why is then tyrosinase capable of reacting with substrates while hemocyanin is not?
Solomon (Angew. Chem. Int. Ed. Engl. 2001, 40, 4570-450):
Difference in accessibility of the active site
Rates of peroxide displacement by azide (measured using UV absorption) at 4°C:
Hemocyanins: k 0.04 h-1
Tyrosinase:
k =0.95 h-1
Hypothesis, 1980:
Solomon et al., JACS 1980, 102, 7339-7344, p.7343
Angew. Chem. Int. Ed. 2001, 40, 4570-4590
Proof, 1998 (J. Biol. Chem. 273, 25889-25892):
Hemocyanine active site*
Phe49 blocks access
to active site
When the N-terminal fragment including Phe49 is removed,
tarantula hemocyanine shows tyrosinase activity
* From X-ray structure of L.polyphemus Hc., Magnus et al., Proteins Struct. Funct.Gen.19, 302-309
Conclusions
In many cases, metalloproteins use the same or similar active site
for different purposes.
The strategies to confer a particular activity to a given site include
- Allowing/disallowing access of substrates to the active site
(including the dynamics of diffusion of substrate/product)
-Modifying the electrostatic potential by mutating the amino acids
coordinated to the metal or surrounding the binding pocket
-Architecture of the binding pocket defines substrate selectivity
and affects energy of transition states→governs reaction outcome