marine worms Brachiopoda Annelida: Magelona papillicornis Iron porphyrin Active site
Monomeric Dinuclear copper Multimeric N. Terwilliger, J. Exp. Biol.201, 10851098 (1998) Dinuclear iron http://notes.chem.usyd.edu.au/course/codd/CHEM3105/Metalloproteins3.pdf
Crystal structure of hemerytrhin in unloaded state (pdb-code 1HMD) Hexacoordinate Fe(II) Pentacoordinate Fe(II) can bind O2 Dinuclear iron active site fixed by a four-helix bundle
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 Bridging carboxylates Extra carboxylates stabilize higher oxidation states 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 Fe IV 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 Cys pKa (SH) = 8.3 neutral at pH 7. Coordination to a metal atom stabilizes anionic form.
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)! 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
Soft ligands (methionine) stabilize higher oxidation states stabilize the lower oxidation state Porphyrins vinyl farnesyl
(isoprenoid chain) methyl formyl Heme a Amino acids/subunit 153
F2 (g) + 2 e = 2 F (aq) MnO4 + 8H+ + 5e = Mn 2+ + 4H2O Cl2 (g) + 2 e = 2 Cl (aq) Pt2+ (aq) + 2 e = Pt (s) Br2 (g) + 2 e = 2 Br (aq) Fe3+ (aq) + e = Fe2+ (aq) I2 (g) + 2 e = 2 I (aq) 2 H2O + O2 (g) + 4 e = 4 OH (aq) O2 + 2H+ + 2e- = H2O2 ronger oxidant
stronger oxidant pH 7) Cu2+ (aq) + 2 e = Cu+ (aq) 2 H+(aq) + 2 e = H2 (g) Fe2+ (aq) + 2 e = Fe (s) Zn2+ (aq) + 2 e = Zn (s) Al3+ (aq) + 3 e = Al (s) Mg2+ (aq) + 2 e = Mg (s) Na+ (aq) + e = Na (s) Li+ (aq) + e =
Li (s) strong reductants + 2.87 + 1.51 + 1.36 + 1.18 + 1.07 + 0.77 + 0.54
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 General rules governing the Redox-potential in a transition-metal complex Larger number of ligands Anionic ligands Soft ligands (methionine) Coordination geometry imposed by the protein
stabilize higher oxidation states stabilize the lower oxidation state can stabilize the higher or the lower oxidation state Hemocyanin: History 1878 Leon Federicq: Sur lhemocyanine, 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 http://webdoc.sub.gwdg.de/diss/2003/ackermann/ackermann.pdf On the search for functional hemocyanin model compounds
Karlin et al., JACS 1988, 110, 36903692 The first model complex showing reversible O2 binding by a dicopper unit However, this complex differs from oxy-Hc: Cu-Cu 1 1
4.36 Oxy-Hc 3.5-3.7 (O-O)[cm-1] 834 744-752 Karlin et al., J. Am. Chem. Soc. 1988, 110, 3690-3692
UV-VIS 440(2000) 525(11500) 590(7600) 1035(160) 340(20000) 580(100) Model complex showing reversible O2 binding and similar features to Hc
Kitajima et al., J. Am. Chem. Soc. 1989, 111, 8975-8976 Cu-Cu 2 3.56 (O-O)[cm-1] 741
Kitajima et al., JACS 1989, 111, 8975-8976 UV-Vis absorption spectra of the oxy forms of hemocyanin and tyrosinase d vd dd 5-9 years later (1994, 1998): Active sites in hemocyanins determined by X-ray crystallography
Magnus et al.,Proteins Struct. Funct. Gen.1994 Limulus polyphemus Cuff et al.,J.Mol.Biol.1998 Octopus dofleini 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 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): 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
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 statesgoverns reaction outcome
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