Metalloproteins reacting with oxygen 1. Why do aerobic organisms need metalloproteins? 2. Oxygen...

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

[email protected]


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.

2s2s

2p 2p

O O2 O

Molecular orbital level diagram for O2: 3g- 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)


xy xz yz

z2 x2-y2

xz

6 ligandsoctahedral

field

5 ligandsoctahedral

field

L

ML L

LL

L

M

L

M

L L

LL



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



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


dxz,dyz

dxy

dz2

dx2-y2

*

*




One of the * orbitals of O2 overlaps with the dz2 orbital of Fe and forms a bond; the other * orbital is non-bonding


1[L5FeO2]

dxz,dyz

dxy

dz2

dx2-y2

*

*



spin inversion


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

2e-2

in vertebrates

Reduction of O2 to H2OCatalyzed by the enzyme

Cytochrome-oxidase

http://www.ul.ie/~childsp/CinA/Issue64/TOC36_Haemoglobin.htm

153 amino acids

2.1. Hemoglobin, Myoglobin & Cytochrome P450

Oxygen binding by myoglobin (Mb)

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-




3. Conclusion

F5351


[email protected]

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

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 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.




3. Conclusion

F5351


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http://notes.chem.usyd.edu.au/course/codd/CHEM3105/Metalloproteins3.pdf

(HOI2)-

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

Amino acids/subunit 153 113 628

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.




3. Conclusion

F5351


[email protected]

Amino acids/subunit 153 113 628

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

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

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+

UV-Vis absorption spectra of the oxy forms of hemocyanin and tyrosinase

d→d

v→d

→d

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

L-DOPAquinone

Syntéza melaninu z tyrosinu katalyzovaná enzymem tyrosináza

http://pollux.chem.umn.edu/~kinsinge/new_homepage/research/gss_presentation_3/sld019.htm





http://pollux.chem.umn.edu/~kinsinge/new_homepage/research/gss_presentation_3/index.htm

http://pollux.chem.umn.edu/~kinsinge/

http://pollux.chem.umn.edu/~kinsinge/new_homepage/research/gss_presentation_3/tsld006.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

Metalloproteins reacting with oxygen 1. Why do aerobic organisms need metalloproteins? 2. Oxygen...

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