Post on 22-Dec-2015
Metalloproteins reacting with oxygen
1. Why do aerobic organisms need metalloproteins?
2. Oxygen transport proteins & Oxygenases2.1. Hemoglobin, Myoglobin & Cytochrome P4502.2. Hemerythrin & Methane monooxygenase2.3. Hemocyanin & Tyrosinase
3. Conclusion
F5351
Jiří Kozelka13.11. 2014
kozelka.jiri@gmail.com
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 G0 = -674 kcal/mol
Elimination of xenobiotics. Example: hydroxalation of hexane by Cytochrome P450OH
OH
OH
+ 1/2 O2
1-Hexanol
3-Hexanol
2-Hexanol
n-Hexane
Cytochrome P450
minor
major
minor
1. The solubility problemWater 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.
Use of oxygen by aerobic organisms is hampered by two problems:
2. The kinetic problemOxygen 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.
Fe2+L
L
L
L
L
OO
Fe2+L
L
L
L
L
OO
Activation of O2 with the help of a transition metal complex:Adduct formation from a pentacoordinated [FeL5]2+ complex and O2
*2p
2p
2pv
*2ph
z
xy
yz
yz
xz
xz
*2pv
2ph
Vazebné a antivazebné molekulové orbitály tvořené atomovými orbitály 2p v molekule O2
antivazebné
vazebné
O O
z
xy
Vazebné a antivazebné molekulové orbitály tvořící vazbu v molekule O2
O O
vazebné
antivazebné
2pv2ph
*2pv*2ph
Fe2+L
L
L
L
L
OO
Fe2+L
L
L
L
L
OO
Activation of O2 with the help of a transition metal complex:Adduct formation from a pentacoordinated [FeL5]2+ complex and O2
Splitting of d orbitals in an octahedral environment(6 equal ligands)
Cetral transition metal atom Lone-pairs of ligands
xy xz yz
z2 x2-y2
L
ML L
LL
L
M
Splitting of d orbitals in an tetragonal environment(5 equal ligands)
Cetral transition metal atom Lone-pairs of ligands
xy xz yz
z2 x2-y2
xz
6 ligandsoctahedral
field
5 ligandsoctahedral
field
L
ML L
LL
L
M
L
M
L L
LL
Splitting of d orbitals in an tetragonal environment(5 equal ligands)
Cetral transition metal atom Lone-pairs of ligands
xy xz yz
z2 x2-y2
xz
x2-y2
z2
6 ligandsoctahedral
field
5 ligandsoctahedral
field
L
ML L
LL
L
M
L
M
L L
LL
Splitting of d orbitals in an tetragonal environment(5 equal ligands)
Cetral transition metal atom Lone-pairs of ligands
xy xz yz
z2 x2-y2
xz
xz xz
xy
x2-y2
z2
6 ligandsoctahedral
field
5 ligandsoctahedral
field
L
ML L
LL
L
M
L
M
L L
LL
3O23[L5FeO2] 1[L5Fe]
dxz,dyz
dxy
dz2
dx2-y2
*
3O2 + 1[L5Fe] 3[L5FeO2]spin-allowed:
n° of unpaired electrons unchanged
(only the two unpairedvalence electrons shown)
Fe2+L
L
L
L
L
Fe2+L
L
L
L
L
3O23[L5FeO2] 1[L5Fe]
dxz,dyz
dxy
dz2
dx2-y2
*
*
3O2 + 1[L5Fe] 3[L5FeO2]spin-allowed:
n° of unpaired electrons unchanged
(only the two unpairedvalence electrons shown)
One of the * orbitals of O2 overlaps with the dz2 orbital of Fe and forms a bond; the other * orbital is non-bonding
3O23[L5FeO2] 1[L5Fe]
1[L5FeO2]
dxz,dyz
dxy
dz2
dx2-y2
*
*
3O2 + 1[L5Fe] 3[L5FeO2]spin-allowed:
n° of unpaired electrons unchanged
spin inversion
(only the two unpairedvalence electrons shown)
process spin-forbiddenbut rendered possibleby spin-orbit coupling
One of the * orbitals of O2 overlaps with the dz2 orbital of Fe and forms a bond; the other * orbital is non-bonding
Fe2+L
L
L
L
L
OO_
__
In transition metal complexes, spin-orbit coupling renders spin-forbidden transitions possible.
Metal complexes can therefore activate (triplet) oxygen for reactionswith (singlet) organic molecules.
[MLn]m+ + 3O21[MLn O2]m+
+ 1[Substrate]1[Oxidation products]
Metal-oxygen adducts can also be usedas oxygen carriers!
2. Oxygen transport proteins & oxygenases
Oxygen transport proteins: O2 binding in active sites
Hemoglobin(vertebrates, some invertebrates)
Hemocyanin(molluscs, some arthropods)
Hemerythrin(some marine invertebrates)
Lippard: Bioinorganic Chemistry, 1994
Aminokyselina histidin tvořící koordinativní vazbu k Fe„proximální histidin“. Toto je jediná kovalentní vazba meziporfyrinem železa a proteinem. Ostatní síly jsou hydrofobní,mezi porfyrinovým cyklem a hydrofobními postrannímiřetězci proteinu.
Fe2+N
N
N
N
N
OO
_
___
Fe3+N
N
N
N
N
OO_
__-_
.O2 oxygen molecule
O2- superoxide anion
http://www.ul.ie/~childsp/CinA/Issue64/TOC36_Haemoglobin.htm
153 amino acids
2.1. Hemoglobin, Myoglobin & Cytochrome P450
Since O2 is a gas, we can replace the concentration [O2] by partial pressure p(O2)
)]([
)(
2
2
OpK
OpY
d
][
)(][
2
2
MbO
OpMbKd
0
20
40
60
80
100
0 30 60 90
Y [%]
p(O2) [Torr]
Cvičení: Jaký význam má směrnice saturační křivky v bodě p(O2) = 0?Znázorněte graficky závislost dY/dp(O2) na p(O2)
p(O2) [Torr] Y [%]0.51235102030405060708090
Cvičení
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.
15.226.341.751.764.178.187.791.593.594.795.596.296.697.0
)]([
)(
2
2
OpK
OpY
d
22
22
2
22 )]([)]([
)(
)]([
1
)( OpK
K
OpK
Op
OpKOdp
dY
d
d
dd
dKOdp
dY 1
)( 2
At p(O2) = 0
Čím silnější afinita mezi Mb a O2, tím strmější křivka v bodě 0;0
Směrnice saturační křivky klesá se stoupajícím tlakem p(O2)
0.00
0.05
0.10
0.15
0.20
0.25
0.30
0 20 40 60 80 100p(O2) [Torr]
dY/dp(O2) [Torr
-1]
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 individualsubunits. Oxygen binding to one subunit of hemoglobin favors the planar format neighboring subunits fully loaded hemoglobin has an affinity similar to thatof 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 groupsof certains amino acids are transformed to carbamate:
amino acidNH2 O C O+
amino acidNH O-
O
+ H+
The liberated H+ protonates histidine residues:
HNN + H+
HNN+ H
At subunit interfaces salt bridges are formed:
amino acidNHO-
O
HNN+ H
These salt bridges favor the domed conformation favor O2 release CO2 favors release of O2 which is then taken up by myoglobin
http://www.chemistry.wustl.edu/~edudev/LabTutorials/Hemoglobin/MetalComplexinBlood.html
In muscles:High CO2 concentration favors domed conformation favors O2 release
In bronchi:Low CO2 concentration favors planar conformation favors O2 binding
FeII O
O
_
__FeIV O
O
_
__
_
_
_ _
Fe(IV)-O22- Fe(II)-O2
0
Fe(III)-O2-
Pauling
Weiss
FeIII O
O
_
__
_
_. .
_
FeIII O
O
_
__
_
..
peroxide
superoxide
dioxygen
Fe(II)-O2, Fe(III)-O2-, or Fe(IV)-O2
2-?
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 O-O [cm-1] d O-O
[A]
O2+ 1905 1.12
O2 1580 1.21
O2- 1097 1.33
O22- 802 1.49
Mb-O2 1105 1.22
M-O2- 1100-1150 1.24-1.31
M- O22- 800-900 1.35-1.50
Oxymyoglobin resembles FeIII-O2-
Metalloproteins reacting with oxygen
1. Why do aerobic organisms need metalloproteins?
2. Oxygen transport proteins & Oxygenases2.1. Hemoglobin, Myoglobin & Cytochrome P4502.2. Hemerythrin & Methane monooxygenase2.3. Hemocyanin & Tyrosinase
3. Conclusion
F5351
Jiří Kozelka13.11. 2014
kozelka.jiri@gmail.com
From: Cécile Claude, „Enzyme Models of Chloroperoxidase and Catalase“, Inaugural Dissertation, Universität Basel, 2001
Hemoproteins: Axial Ligands and Functions
Hemoprotein proximal ligand 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
Source: C. Capeillere-Blandin, D. Matthieu & D. Mansuy,Biochem. J. 2005, 392, 583-587
Modification of the FeII/FeIII redox potential by the protein environment
Strong reductants
Strong oxidants FeII (Red.) stable
FeIII (Ox.) stable
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.
antibiotic
local anesthetic steroid hormone
carcinogen from fungi
Alkaloid from Taxus brevifolia, potent anti-cancer drug
Examples of Cytochrome P450 substrates
Hydroxylation at:-aliphatic carbons
-aromatic carbons-double bonds-heteroatoms
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 oxidačně-redukční 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.
Metalloproteins reacting with oxygen
1. Why do aerobic organisms need metalloproteins?
2. Oxygen transport proteins & Oxygenases2.1. Hemoglobin, Myoglobin & Cytochrome P4502.2. Hemerythrin & Methane monooxygenase2.3. Hemocyanin & Tyrosinase
3. Conclusion
F5351
Jiří Kozelka13.11. 2014
kozelka.jiri@gmail.com
Crystal structure of hemerytrhin in unloaded state (pdb-code 1HMD)
Dinuclear iron active site fixed by a four-helix bundle
Hexacoordinate Fe(II)
Pentacoordinate Fe(II)
can bind O2
Sipuncula Priapulida Brachiopoda
Magelona papillicornis
Hemerythrin je metaloprotein transportující kyslík u některých bezobratlých
Active sites of the reduced forms of Hemerythrin, Ribonucleotide Reductase R2 protein, the hydroxylase component of Methane Monooxygenase, and 9 desaturase
Bridging carboxylates
Extra carboxylates stabilize higher oxidation states
Catalytic Cycle of soluble Methane Monooxygenase (sMMO)
Kopp & Lippard, Current Op. Chem. Biol. 2002, 568
Methane is a very unreactive compound. Needs an extremely strong oxydant to be hydroxylated.
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.
Metalloproteins reacting with oxygen
1. Why do aerobic organisms need metalloproteins?
2. Oxygen transport proteins & Oxygenases2.1. Hemoglobin, Myoglobin & Cytochrome P4502.2. Hemerythrin & Methane monooxygenase2.3. Hemocyanin & Tyrosinase
3. Conclusion
F5351
Jiří Kozelka13.11. 2014
kozelka.jiri@gmail.com
Megathura crenulata
Octopus dofleiniPanulirus interruptus
Linulus polyphemus
Hemocyanin je metaloprotein transportující kyslík u většiny měkkýšů a u některých korýšů
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 HaemocyaninsZ. Physiol. Chem. 33, 370Hemocyanin contains copper
1940 W. A. Rawlinson, Australian J. Exp. Biol. Med. Sci. 18,131Oxy-hemocyanin is diamagnetic
http://webdoc.sub.gwdg.de/diss/2003/ackermann/ackermann.pdf
Známé a hypotetické (*) komplexy mědi s jednotkou O2
The first model complex showing reversible O2 binding by a dicopper unit
Karlin et al., J. Am. Chem. Soc. 1988, 110, 3690-3692
However, this complex differs from oxy-Hc:
Cu-Cu[Å] υ(O-O)[cm-1] UV-VIS
1 4.36 834 440(2000) 525(11500)
590(7600) 1035(160)
Oxy-Hc 3.5-3.7 744-752 340(20000) 580(100)
1
Model complex showing reversible O2 binding and similar features to Hc
Cu-Cu[Å] υ(O-O)[cm-1] UV-VIS
3.56 741 349(21000) 551(790)
3.5-3.7 744-752 340(20000) 580(100)
2
2
Oxy-Hc
Kitajima et al., J. Am. Chem. Soc. 1989, 111, 8975-8976
Kitajima et al., JACS 1989, 111, 8975-8976Karlin et al., JACS 1988, 110, 3690’3692
[Cu{HB(3,5-iPr2pz)3}]2(O2)
Functional hemocyanin models
[(tmpa)2Cu2O2]2+
5-9 years later (1994, 1998):Active sites in hemocyanins determined by X-ray crystallography
Limulus polyphemus Octopus dofleini
Magnus et al.,Proteins Struct. Funct. Gen.1994 Cuff et al.,J.Mol.Biol.1998
An earlier model for hemocyanin...
…turned out to be a model for the enzyme tyrosinase!
Karlin et al., JACS 1984, 106, 2121-2128
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
Solomon et al., JACS 1980, 102, 7339-7344, p.7343Angew. Chem. Int. Ed. 2001, 40, 4570-4590
Hypothesis, 1980:
Proof, 1998 (J. Biol. Chem. 273, 25889-25892):
Hemocyanine active site*
Phe49 blocks accessto 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