Amino acids/subunit 153 113 628

Sipuncula Priapulida

Brachiopoda

Annelida: Magelona papillicornis

marine worms

Active site Iron porphyrin Dinuclear copperDinuclear

iron

Monomeric Multimeric

N. Terwilliger, J. Exp. Biol.201, 1085–1098 (1998)

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

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

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

Active sites of the reduced forms of Hemerythrin, Ribonucleotide Reductase R2 protein, and the hydroxylase component of Methane Monooxygenase

Extra carboxylates stabilize higher oxidation states

Bridging carboxylates

Catalytic Cycle of soluble Methane Monooxygenase (sMMO)

Kopp & Lippard, Current Op. Chem. Biol. 2002, 568

Remember:

Hr and sMMO share the main features:a four-helix-bundle surrounding a Fe-(carboxylato)2-Fe core

but differ in the particular environment of the Fe centers:

-Hr coordination sphere is more histidine rich-Hr permits only terminal O2-coordination to a single iron, while sMMO diiron center presents open or labile coordination sites on both Fe-sMMO shows much greater coordinative flexibility upon oxidation-The larger number of anionic ligands allows sMMO to achieve the FeIV oxidation state needed for oxidation methane.

Intermezzo: Bioligands

Histidin

pKa (His+) = 6.0 neutral at pH 7, but can be easily protonated, can serve as „proton shuttle“Both tautomers are found as ligands

pKa (His) = 14.4 rarely exists in deprotonated form as bridging ligand (in Cu-Zn superoxide-dismutase)

Aspartate & Glutamate

pKa (COOH) = 3.9 pKa (COOH) = 4.1 at pH 7 anionic even without coordination to a metal atom

Cysteinate

pKa (SH) = 8.3 neutral at pH 7. Coordination to a metal atom stabilizes anionic form.

Cys

Tyrosinate

pKa (TyrH) = 10.1 neutral at pH 7. Coordination to a metal atom stabilizes anionic form.Can be oxidized to a radical Tyr· (see RNR-R2)!

Tyr

Intermezzo: Bioligands

Methionine

neutral, „soft“ ligand

prefers FeII to FeIII

occurs in cytochromes (electron transfer proteins) where it stabilizes the lower oxidation state

General rules governing the Redox-potential in a transition-metal complex

Larger number of ligands

Anionic ligands stabilize higher oxidation states

Soft ligands (methionine) stabilize the lower oxidation state

Porphyrins

Heme a

vinyl farnesyl (isoprenoid chain)

methyl

formyl

Amino acids/subunit 153 113 628

Megathura crenulata

Octopus dofleiniPanulirus interruptus

Linulus polyphemus

Chemistry enabling O2 transport by hemocyanin

2Cu+ + O2 2Cu2+ + O22-

Red. Ox. Ox. Red.

Loading O2:

Unoading O2:

2Cu2+ + O22- 2Cu+ + O2

Ox. Red. Red. Ox.

Vybrané standardní redukční potenciály při 25°C:F2 (g) + 2 e– = 2 F– (aq) + 2.87

MnO4 – + 8H+ + 5e– = Mn 2+ + 4H2O + 1.51

Cl2 (g) + 2 e– = 2 Cl– (aq) + 1.36

Pt2+ (aq) + 2 e– = Pt (s) + 1.18Br2 (g) + 2 e– = 2 Br– (aq) + 1.07

Fe3+ (aq) + e– = Fe2+ (aq) + 0.77I2 (g) + 2 e– = 2 I– (aq) + 0.54

2 H2O + O2 (g) + 4 e– = 4 OH– (aq) + 0.41

O2 + 2H+ + 2e- = H2O2 + 0.35 (at pH 7)

Cu2+ (aq) + 2 e– = Cu+ (aq) + 0.15 2 H+(aq) + 2 e– = H2 (g) 0.00

Fe2+ (aq) + 2 e– = Fe (s) - 0.45Zn2+ (aq) + 2 e– = Zn (s) - 0.76Al3+ (aq) + 3 e– = Al (s) - 1.67Mg2+ (aq) + 2 e– = Mg (s) - 2.37Na+ (aq) + e– = Na (s) - 2.71Li+ (aq) + e– = Li (s) - 3.04

strong oxidants

strong reductants

stronger oxidant stronger oxidant

Chemistry enabling O2 transport by hemocyanin

2Cu+ + O2 2Cu2+ + O22-

Red. Ox. Ox. Red.

Loading O2:

Unloading O2:

2Cu2+ + O22- 2Cu+ + O2

Ox. Red. Red. Ox.

O2 stronger oxidant

Cu+ stronger reductant OK

would procede in reverse directionin aqueous solutions at pH 7

But: Tetrahedral Cu- environment in hemocyanin favors Cu+ !

The potential of the Cu 2+/Cu+ couple shifts to 0.3-0.4 V The potentials of both half-reactions become similar The whole reaction becomes reversible

General rules governing the Redox-potential in a transition-metal complex

Larger number of ligands

Anionic ligands stabilize higher oxidation states

Coordination geometry can stabilize the higher or the lower oxidation stateimposed by the protein

Soft ligands (methionine) stabilize the lower oxidation state

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

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

                                                                                                         

               

         

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

L-DOPAquinone

The enzyme tyrosinase catalyzes the synthesis of the pigment melanin from tyrosine

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

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

An earlier model for hemocyanin...

…turned out to be a model for the enzyme tyrosinase!

Karlin et al., JACS 1984, 106, 2121-2128

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