1. Amino acids/subunit 153 113 628

2. Sipuncula Priapulida Brachiopoda Annelida: Magelona papillicornis marine worms

3. Active site Iron porphyrin Dinuclear copper Dinuclear iron Monomeric Multimeric N. Terwilliger, J. Exp. Biol.201, 1085–1098 (1998)

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

5. 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 O 2

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

8. 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

9. Catalytic Cycle of soluble Methane Monooxygenase (sMMO) Kopp & Lippard, Current Op. Chem. Biol. 2002, 568

10. 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 O 2 -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 Fe IV oxidation state needed for oxidation methane.

11. Intermezzo: Bioligands Histidin pK a (His + ) = 6.0  neutral at pH 7, but can be easily protonated, can serve as „proton shuttle“ Both tautomers are found as ligands pK a (His) = 14.4  rarely exists in deprotonated form as bridging ligand (in Cu-Zn superoxide-dismutase)

12. Aspartate & Glutamate pK a (COOH) = 3.9 pK a (COOH) = 4.1  at pH 7 anionic even without coordination to a metal atom

13. Cysteinate pK a (SH) = 8.3  neutral at pH 7. Coordination to a metal atom stabilizes anionic form. Cys

14. Tyrosinate pK a (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

15. Intermezzo: Bioligands Methionine neutral, „soft“ ligand  prefers Fe II to Fe III occurs in cytochromes (electron transfer proteins) where it stabilizes the lower oxidation state

16. 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

17. Porphyrins Heme a vinyl  farnesyl (isoprenoid chain) methyl  formyl

18. Amino acids/subunit 153 113 628

19. Megathura crenulata Octopus dofleini Panulirus interruptus Linulus polyphemus

21. Chemistry enabling O 2 transport by hemocyanin 2Cu + + O 2  2Cu 2+ + O 2 2- Red. Ox. Ox. Red. Loading O 2 : Unoading O 2 : 2Cu 2+ + O 2 2-  2Cu + + O 2 Ox. Red. Red. Ox.

22. Vybrané standardní redukční potenciály při 25°C: F 2 (g) + 2 e – = 2 F – (aq) + 2.87 MnO 4 – + 8H + + 5e – = Mn 2+ + 4H 2 O+ 1.51 Cl 2 (g) + 2 e – = 2 Cl – (aq) + 1.36 Pt 2+ (aq) + 2 e– = Pt (s) + 1.18 Br 2 (g) + 2 e – = 2 Br – (aq) + 1.07 Fe 3+ (aq) + e – = Fe 2+ (aq) + 0.77 I 2 (g) + 2 e – = 2 I – (aq) + 0.54 2 H 2 O + O 2 (g) + 4 e – = 4 OH – (aq)+ 0.41 O 2 + 2H + + 2e - = H 2 O 2 + 0.35 (at pH 7) Cu 2+ (aq) + 2 e – = Cu + (aq) + 0.15 2 H + (aq) + 2 e – = H 2 (g) 0.00 Fe 2+ (aq) + 2 e – = Fe (s) - 0.45 Zn 2+ (aq) + 2 e – = Zn (s) - 0.76 Al 3+ (aq) + 3 e – = Al (s) - 1.67 Mg 2+ (aq) + 2 e – = Mg (s) - 2.37 Na + (aq) + e – = Na (s) - 2.71 Li + (aq) + e – = Li (s) - 3.04 strong oxidants strong reductants stronger oxidant

23. Chemistry enabling O 2 transport by hemocyanin 2Cu + + O 2  2Cu 2+ + O 2 2- Red. Ox. Ox. Red. Loading O 2 : Unloading O 2 : 2Cu 2+ + O 2 2-  2Cu + + O 2 Ox. Red. Red. Ox. O 2 stronger oxidant Cu + stronger reductant  OK  would procede in reverse direction in 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

24. General rules governing the Redox-potential in a transition-metal complex Larger number of ligands Anionic ligands stabilize higher oxidation states Coordination geometrycan stabilize the higher or the lower oxidation state imposed by the protein Soft ligands (methionine) stabilize the lower oxidation state

25. 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

26. http://webdoc.sub.gwdg.de/diss/2003/ackermann/ackermann.pdf

27. On the search for functional hemocyanin model compounds Karlin et al., JACS 1988, 110, 3690’3692

28. The first model complex showing reversible O 2 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 14.36 834 440(2000) 525(11500) 590(7600) 1035(160) Oxy-Hc3.5-3.7 744-752 340(20000) 580(100) 1

29. Model complex showing reversible O 2 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

30. Kitajima et al., JACS 1989, 111, 8975-8976 Karlin et al., JACS 1988, 110, 3690’3692 [Cu{HB(3,5-iPr 2 pz) 3 }] 2 (O 2 ) Functional hemocyanin models [(tmpa) 2 Cu 2 O 2 ] 2 +

31. UV-Vis absorption spectra of the oxy forms of hemocyanin and tyrosinase d→dd→d  v →d   →d

32. 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

33. Slide 6 of 21 http://pollux.chem.umn.edu/~kinsinge/new_homepage/research/gss_presentation_3/sld019.htm

34. L-DOPAquinone The enzyme tyrosinase catalyzes the synthesis of the pigment melanin from tyrosine

35. 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

36. Solomon et al., JACS 1980, 102, 7339-7344, p.7343 Angew. Chem. Int. Ed. 2001, 40, 4570-4590 Hypothesis, 1980: Proof, 1998 (J. Biol. Chem. 273, 25889-25892):

37. 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

38. An earlier model for hemocyanin... …turned out to be a model for the enzyme tyrosinase! Karlin et al., JACS 1984, 106, 2121-2128

39. 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