Metal%20Ions%20in%20Biological%20Systems

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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
Jirí Kozelka 13.11. 2014 kozelka.jiri_at_gmail.com
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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
minor
minor
major
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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.
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Molecular orbital level diagram for O2 3Sg- state
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Activation of O2 with the help of a transition
metal complex Adduct formation from a
pentacoordinated FeL52 complex and O2
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Vazebné a antivazebné molekulové orbitály tvorené
atomovými orbitály 2p v molekule O2
s2p
p2ph
antivazebné
xz
p2pv
yz
p2ph
xz
vazebné
p2pv
yz
Index h horizontální Index v vertikální
s2p
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Vazebné a antivazebné molekulové orbitály tvorící
vazbu p v molekule O2 prostorové usporádání
p2ph
p2pv
antivazebné
vazebné
p2ph
O
p2pv
O
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Activation of O2 with the help of a transition
metal complex Adduct formation from a
pentacoordinated FeL52 complex and O2
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Splitting of d orbitals in an octahedral
environment (6 equal ligands)
Cetral transition metal atom
Lone-pairs of ligands
6 ligands octahedral field
M
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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
xz
M
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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
x2-y2
z2
xz
M
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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
x2-y2
z2
xz
xy
xz
M
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3O2 1L5Fe ? 3L5FeO2
spin-allowed n of unpaired electrons unchanged
(only the two unpaired valence electrons shown)
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3O2 1L5Fe ? 3L5FeO2
spin-allowed n of unpaired electrons unchanged
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
(only the two unpaired valence electrons shown)
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3O2 1L5Fe ? 3L5FeO2
spin-allowed n of unpaired electrons unchanged
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
spin inversion
process spin-forbidden but rendered possible by
spin-orbit coupling
(only the two unpaired valence electrons shown)
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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.
1Substrate
1MLn O2m
MLnm 3O2
1Oxidation products
Metal-oxygen adducts can also be used as oxygen
carriers!
2. Oxygen transport proteins oxygenases
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Oxygen transport proteins O2 binding in active
sites
Hemoglobin (vertebrates, some invertebrates)
Hemocyanin (molluscs, some arthropods)
Hemerythrin (some marine invertebrates)
Lippard Bioinorganic Chemistry, 1994
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O2 oxygen molecule O2- superoxide anion
Aminokyselina histidin tvorící 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 retezci proteinu.
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in vertebrates
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2e-
Reduction of O2 to H2O Catalyzed by the
enzyme Cytochrome-oxidase
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2.1. Hemoglobin, Myoglobin Cytochrome P450
153 amino acids
http//www.ul.ie/childsp/CinA/Issue64/TOC36_Haemo
globin.htm
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Vazba myoglobinu (Mb) na kyslík
Cvicení 1 definujte rovnovážnou konstantu pro
zpetnou reakci (tzv. disociacní konstantu, Kd)
Cvicení 2 definujte saturaci vazebných míst, Y,
definovanou rovnicí dole, pomocí Kd a O2 jako
promenných. Nahradte ve vzorcích pro Kd a pro Y
koncentraci O2 parciálním tlakem p(O2).
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Cvicení 3 Vypoctete krivku frakcní saturace
kyslíku na myoglobinu. Disociacní konstanta
komplexu MbO2 je, pri 37 C, pH 7 a p 760
Torr, Kd 2.8 Torr.
p(O2) Torr Y 0.5 1 2 3 5 10 20 30 40 50 6
0 70 80 90
Cvicení 4 Jaký význam má smernice saturacní
krivky v bode p(O2) 0? Znázornete graficky
závislost dY/dp(O2) na p(O2)
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• 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/LabTutorial
s/Hemoglobin/MetalComplexinBlood.html
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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
The liberated H protonates histidine residues
At subunit interfaces salt bridges are formed
These salt bridges favor the domed conformation ?
favor O2 release ? CO2 favors release of O2 which
is then taken up by myoglobin
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In bronchi Low CO2 concentration favors planar
conformation ? favors O2 binding
In muscles High CO2 concentration favors domed
conformation ? favors O2 release
http//www.chemistry.wustl.edu/edudev/LabTutorial
s/Hemoglobin/MetalComplexinBlood.html
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Fe(II)-O2, Fe(III)-O2-, or Fe(IV)-O22-?
What experimental data can be used to determine
whether oxygen in oxyhemoglobin resembles more
to Fe(III)-O2- or to Fe(II)-O2?
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Stretching frequencies and bond lengths in
dioxygen species
Species nO-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
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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
Jirí Kozelka 13.11. 2014 kozelka.jiri_at_gmail.com
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Hemoproteins Axial Ligands and Functions
From Cécile Claude, Enzyme Models of
Chloroperoxidase and Catalase, Inaugural
Dissertation, Universität Basel, 2001
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Modification of the FeII/FeIII redox potential by
the protein environment
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
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.
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Examples of Cytochrome P450 substrates

• Hydroxylation at
• aliphatic carbons

• -aromatic carbons
• double bonds

steroid hormone
local anesthetic
-heteroatoms
carcinogen from fungi
antibiotic
Alkaloid from Taxus brevifolia, potent
anti-cancer drug
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Cytochrome P450cam (Campher-5-monooxygenase
pdb-code 1T86)
access for substrate and O2
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• Hlavní dva rozdíly mezi hemoproteiny myoglobin a
  cytochrom P450, duležité pro jejich ruzné funkce
• Prístupový kanál vedoucí ke kofaktoru (hemu) je u
  myoglobinu velmi úzký, nedovoluje prístup vetším
  molekulám než O2. U cytochromu P450 je kanál
  širší a v blízkosti kofaktoru obsahuje místo s
  vysokou afinitou pro specifické substráty.
• Distální cystein a okolí kofaktoru snižuje u
  cytochromu P450 oxidacne-redukcní potenciál Fe,
  takže tento metaloprotein muže fungovat jako
  oxygenáza a Fe v katalytickém cyklu muže
  krátkodobe existovat v oxidacním stupni Fe(V).
  Tento velmi reaktivní prechodný stav je schopen
  hydroxylovat i pomerne nereaktivní alifatické
  atomy uhlíku.

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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
Jirí Kozelka 13.11. 2014 kozelka.jiri_at_gmail.com
37
http//notes.chem.usyd.edu.au/course/codd/CHEM3105
/Metalloproteins3.pdf
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Crystal structure of hemerytrhin in unloaded
state (pdb-code 1HMD)
Dinuclear iron active site fixed by a four-helix
bundle
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Amino acids/subunit 153 113
628
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Sipuncula
Priapulida
Brachiopoda
Hemerythrin je metaloprotein transportující
kyslík u nekterých bezobratlých
Magelona papillicornis
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Active sites of the reduced forms of Hemerythrin,
Ribonucleotide Reductase R2 protein, the
hydroxylase component of Methane Monooxygenase,
and D9 desaturase
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Catalytic Cycle of soluble Methane Monooxygenase
(sMMO)
Kopp Lippard, Current Op. Chem. Biol. 2002, 568
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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
Jirí Kozelka 13.11. 2014 kozelka.jiri_at_gmail.com
45
Amino acids/subunit 153 113
628
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Hemocyanin je metaloprotein transportující kyslík
u vetšiny mekkýšu a u nekterých korýšu
Panulirus interruptus
Octopus dofleini
Megathura crenulata
Linulus polyphemus
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• Hemocyanin History
• Leon Federicq Sur lhemocyanine, substance
  nouvelle
• de sang de Poulpe (Octopus vulgaris)
• (Compt. Rend. Acad. Sci. 87, 996-998)
• Discovery
• M. Henze Zur Kenntniss des Haemocyanins
• Z. Physiol. Chem. 33, 370
• Hemocyanin contains copper
• W. A. Rawlinson, Australian J. Exp. Biol. Med.
  Sci. 18,
• 131
• Oxy-hemocyanin is diamagnetic

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Známé a hypotetické () komplexy medi s jednotkou
O2
http//webdoc.sub.gwdg.de/diss/2003/ackermann/acke
rmann.pdf
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On the search for functional hemocyanin model
compounds
Karlin et al., JACS 1988, 110, 36903692
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The first model complex showing reversible O2
binding by a dicopper unit
However, this complex differs from
oxy-Hc Cu-CuÅ ?(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
Karlin et al., J. Am. Chem. Soc. 1988, 110,
3690-3692
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Model complex showing reversible O2 binding and
similar features to Hc
Kitajima et al., J. Am. Chem. Soc. 1989, 111,
8975-8976
Cu-CuÅ ?(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
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Functional hemocyanin models
(tmpa)2Cu2O22
CuHB(3,5-iPr2pz)32(O2)
Kitajima et al., JACS 1989, 111, 8975-8976
Karlin et al., JACS 1988, 110, 36903692
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UV-Vis absorption spectra of the oxy forms of
hemocyanin and tyrosinase
ps?d
pv?d
d?d
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5-9 years later (1994, 1998) Active sites in
hemocyanins determined by X-ray crystallography
Magnus et al.,Proteins Struct. Funct. Gen.1994
Cuff et al.,J.Mol.Biol.1998
Limulus polyphemus
Octopus dofleini
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An earlier model for hemocyanin...
turned out to be a model for the enzyme
tyrosinase!
Karlin et al., JACS 1984, 106, 2121-2128
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L-DOPAquinone
Syntéza melaninu z tyrosinu katalyzovaná enzymem
tyrosináza
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http//pollux.chem.umn.edu/kinsinge/new_homepage/
research/gss_presentation_3/sld019.htm
Slide 6 of 21
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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 4C Hemocyanins k 0.04
h-1 Tyrosinase k 0.95 h-1
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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)
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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
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• 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