Metalloproteins and the Chemistry of Oxygen Binding

1. Amino acids/subunit 153 113 628

2. Sipuncula Priapulida marine worms Brachiopoda Annelida: Magelona papillicornis

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

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

5. Hexacoordinate Fe(II) Pentacoordinate Fe(II) can bind O2 Crystal structure of hemerytrhin in unloaded state (pdb-code 1HMD) Dinuclear iron active site fixed by a four-helix bundle

6. http://notes.chem.usyd.edu.au/course/codd/CHEM3105/Metalloproteins3.pdfhttp://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 Bridging carboxylates Extra carboxylates stabilize higher oxidation states

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 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.

11. 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)

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

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

14. Tyrosinate Tyr • 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)!

15. Intermezzo: Bioligands Methionine neutral, „soft“ ligand • prefers FeII to FeIII 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 vinyl  farnesyl (isoprenoid chain) methyl  formyl Heme a

18. Amino acids/subunit 153 113 628

19. Panulirus interruptus Octopus dofleini Linulus polyphemus Megathura crenulata

21. Chemistry enabling O2 transport by hemocyanin Loading O2: 2Cu+ + O2 2Cu2+ + O22- Red. Ox. Ox. Red. Unoading O2: 2Cu2+ + O22- 2Cu+ + O2 Ox. Red. Red.Ox.

22. strong oxidants 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) +1.18 Br2 (g) + 2 e– = 2 Br– (aq) + 1.07 Fe3+ (aq) + e– = Fe2+ (aq) + 0.77 I2 (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) - 0.45 Zn2+ (aq) + 2 e– = Zn(s) - 0.76 Al3+ (aq) + 3 e– = Al(s) - 1.67 Mg2+ (aq) + 2 e– = Mg(s) - 2.37 Na+ (aq) + e– = Na(s) - 2.71 Li+ (aq) + e– = Li(s) - 3.04 stronger oxidant stronger oxidant strong reductants

23. Chemistry enabling O2 transport by hemocyanin O2 stronger oxidant Cu+ stronger reductant Loading O2:  OK 2Cu+ + O2 2Cu2+ + O22- Red. Ox. Ox. Red. Unloading O2: 2Cu2+ + O22- 2Cu+ + O2 Ox. Red. Red.Ox. • 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 Soft ligands (methionine) stabilize the lower oxidation state Coordination geometry can stabilize the higher or the lower oxidation state imposed by the protein

25. Hemocyanin: History • Leon Federicq: Sur l‘hemocyanine, 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

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 O2 binding by a dicopper unit 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 Karlin et al., J. Am. Chem. Soc.1988,110, 3690-3692

29. Model complex showing reversible O2 binding and similar features to Hc Kitajima et al., J. Am. Chem. Soc.1989,111, 8975-8976 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

30. Functional hemocyanin models [(tmpa)2Cu2O2]2+ [Cu{HB(3,5-iPr2pz)3}]2(O2) Karlin et al., JACS 1988, 110, 3690’3692 Kitajima et al., JACS 1989, 111, 8975-8976

31. UV-Vis absorption spectra of the oxy forms of hemocyanin and tyrosinase ps→d pv→d d→d

32. 5-9 years later (1994, 1998): Active sites in hemocyanins determined by X-ray crystallography Cuff et al.,J.Mol.Biol.1998 Magnus et al.,Proteins Struct. Funct. Gen.1994 Limulus polyphemus Octopus dofleini

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

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. 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):

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