Cobre – Proteínas Y Enzimas

CURSO QUÍMICA BIOINORGÁNICA

UNAM Octobre 15/17, 2012 PETER M.H. KRONECK, Lab 212

Cobre – Proteínas y Enzimas

Cuprum, from Cyprium – Metal of Cyprus

Bo G. Malmström, Göteborg, Hans Freeman, Sidney, 19291- 2008 1927-2000 Cobre – Metal importante en Biología y Quimíca

• Laccase (Keilin & Mann, Nature, 1939) • Blue color and EPR of laccase (Malmström, et al., Nature, 1959) • L. Gehrig‘s disease/SOD (McCabe et al., PNAS, 1995) • CuA in Cytochrome c oxidase (Tsukihara et al., Science, 1996) • Fe uptake (Stearman et al., Science, 1996) • A Mo-S-Cu Cluster in CODH (Dobbek et al., PNAS, 2002) • Bioinorganic Chemistry in the postgenomic era; Cu trafficking (Bertini and Rosato, PNAS, 2003) • Metals in Neurobiology: Probing Their Chemistry and Biology with Molecular Imaging (Que et al., Chem. Rev, 2008) • Copper Transport in Mammalian Cells: Special Care for a Metal with Special Needs (Kaplan and Lutsenko, JBiolChem, 2009) • Zeroing in on a new copper site (Rosenzweig, Nature, 2009)

2 Artículos que introducen

R. Malkin and B.G. Malmström (1970) Adv. Enzymol. 33, 177-244, The state and function of copper in biological systems.

R. Basosi, W.E. Antholine, and J.S. Hyde (1993) Biological Magnetic Resonance, 13, 103 - 150, Multifrequency ESR of Copper. Biophysical Applications.

B.G. Malmström and J. Leckner (1998) Curr. Op. Chem. Biol. 2, 286-292 The chemical biology of copper.

Chemical Reviews (1996) and (2004) Bioinorganic Enzymology with preface by R.H. Holm and E.I. Solomon 96, and Biomimetic Inorganic Chemistry with preface by R.H. Holm and E.I. Solomon 104

Handbook of metalloproteins (A. Messerschmidt, T. L. Poulos, K. Wieghardt, R. Huber, eds), Wiley, 2001, 2004, 2009 on-line edition

J.H. Kaplan and S. Lutsenko (2009) J. Biol. Chem. 284, 25461-25465, Copper Transport in Mammalian Cells: Special Care for Metal with Special Needs 3 Pioneros de Cu Química Bioinorgánica Helmut Beinert (1995) Crystals and structures of cytochrome c oxidases – the end of an arduous road Chem. Biol., 2, 781-785 Bo G. Malmström (1997) A life with the metals of life Selected Topics in the History of Biochemistry Comprehensive Biochemistry, 40, 277-331 Edward I. Solomon (2006) Spectroscopic Methods in Bioinorganic Chemistry: Blue to Green to Red Copper Sites Inorg. Chem. , 45, 8012-8025 William B. Tolman (2006) Using synthetic chemistry to understand copper protein active sites: a personal perspective J. Biol. Chem., 11, 261-271 Yi Lu (2006) Biosynthetic Inorganic Chemistry Angew. Chem. Int. Ed. 2006, 45, 5588 – 5601 Kenneth D. Karlin (2007) Heme-copper/dioxygen adduct formation, properties, and reactivity. Accounts of Chemical Research, 40, 563-572. 4 Objetivo: De Estructuras cristalinas a Orbitales moleculares para Funcionar

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Edward I. Solomon, Stanford 

0 25000 20000 15000 10000 Wavenumber (cm-1) EPR

2800 3000 3200 3400 Magnetic field (G)

5 H Los elementos de vida He www.webelements.com Li Be B C N O F Ne

Na Mg Al Si P S Cl Ar

K Ca Sc Ti V Cr Mn Fe Co Ni Cu Zn Ga Ge As Se Br Kr

Rb Sr Y Zr Nb Mo Tc Ru Rh Pd Ag Cd In Sn Sb Te I Xe

Cs Ba La Hf Ta W Re Os Ir Pt Au Hg Tl Pb Bi Po At Rn

Abundancia en el cuerpo (75 kg) Ca: 1.2 kg K: 150 g Fe: 4-7 g S: 140 g Na: 70 g Zn: 2-3 g P: 780 g Mg: 20-30 g Cu: 70-100 mg Mn: 10 mg 6 Formas de Vida – De Anaerobio a Aerobio condiciones anóxicas (-O2) contra condiciones óxicas (+O2)

7 Cu y O2 – Una estrecha relación

133 pm sp * O - 1145 cm-1 -0.33V 2 p* +0.94V O2 112 pm 149 pm sp -1 1554 cm-1 H2O2 842 cm p +0.38V s • * s OH +H2O

+2.31V

3 + S 2H8 2O E‘o vs NHE, pH 7.25 Propiedades de Cu en sus estados de oxidación diferentes (1)

29Cu, (Ar) 3d104s1

most common oxidation numbers +1/+2; stable Cu(III) peptide complexes

coordination numbers/geometries: Cu2+ 4,5,6; Cu+ 2,3,4

ionic radii/oxidation state/coordination number: 57pm/+2/4; 46pm/+1/2, 60pm/+1/4

redox potential Cu2+/Cu 0.342 V

isotopes: 63(69.2%); 65 (30.8%); 64 (ß-, 13 h)

9 Propiedades de Cu en sus estados de oxidación diferentes (2)

nuclear spin I: 3/2 (63, 65), 1 (64)

large nuclear quadrupole moment

magnetic moment µeff: 1.73 B.M. (Cu2+; expt. 1.8 - 2.1 B.M.)

among the divalent ions of the 3d elements Cu2+ forms the most stable complexes (Irving-Williams)

classification HSAB hard (Cu2+), soft (Cu+)

fast ligand exchange rate

10 Color y Magnetiso  Técnicas de espectroscopia

UV/VIS/near IR spectroscopy/resonance Raman

Magnetic resonance (CW and pulsed EPR, multifrequency EPR, ENDOR, NMR)

Fluorescence/luminescence spectroscopy

Circular dichroism (CD), magnetic circular dichroism (MCD) spectroscopy

X-ray absorption spectroscopy/XAS/EXAFS

Magnetic susceptibility

Fast kinetic methods (stopped-flow, rapid quench, pulse radiolysis, flash photolysis)

11 Proteína Ligantes – Residuos de Aminoácido N O S

Tyr

His Cys

Glu(+Asp)

Met Lys Ser 12 Clases de Proteínas de Cobre

Classification according to function electron transfer (ET), O2 transport; activation of small inorganic - molecules, such as O2, NO2 , N2O, CO; Fe metabolism; red/ox of complex organic substrates

Multi-copper enzymes catechol oxidase, tyrosinase, laccase, ascorbate oxidase, ceruloplasmin, nitrite reductase, nitrous oxide reductase, cytochrome c oxidase, CO dehydrogenase, particulate methane monooxygenase

Cu homeostasis Cu-ATPases, Cu chaperones

13 Funciones de Proteínas de Cobre

• Cu homeostasis (Cu ATPases, chaperones) • Fe homeostasis (FET multi-copper oxidase)

• Binding of O2 • Electron transfer

• Activation of inert molecules (O2, CO, CH4, N2O) • Red/ox of in/organic molecules (amines, quercetin, ascorbate, .- - catechol, heme degradation products; O2 , NO2 ) • Red/ox of metals (metal oxides as substrates for bacteria)

14 Tipos de cobre

Concept of Malkin and Malmström (1971) Classification of Cu sites according to UV/VIS and EPR Properties Type 1 (blue; 1), 2 (non-blue; 1), 3 (EPR-silent; 2)

Blumberg-Peisach Plot (gII vs AII) CuA (mixed-valence, [Cu(1.5+...Cu(1.5+)]) CuZ (4-Cu-1-Sulfide Cluster)

Mo-Sulfide-Cu Cluster 15 Aspectos estructurales de Sitios de Cobre

Type 1 site: plastocyanin, ascorbic acid oxidase Type 2 site: superoxide dismutase, galactose oxidase Type 3 site: hemocyanin, ascorbic acid oxidase Mixed-valence & exchange-coupled sites: nitrous oxide reductase, cytochrome c oxidase, CO dehydrogenase CuZ site: nitrous oxide reductase Cu active site in pMMO – binuclear ?? Role of Fe ?

Tipos de cobre

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Concept of Malkin and Malmström (1971) Classification of Cu sites according to UV/Vis and EPR Properties Type 1 (blue; mononuclear)

17 Plastocyanin: Blue Type 1 Cu Sitio Función: Proteína de Transferencia electrónica (Fotosíntesis)

Covalent Cu-Cys p-bond is mainly responsible for its unique properties EI Solomon, Inorg. Chem. 2006, 45, 8012-8025

41% Cu 38-45% S PDB Code: 1PLC HC Freeman, 1978 Cu(II) Spin-Distribution 18 Edward I. Solomon (2006) Spectroscopic Methods in Bioinorganic Chemistry: Blue to Green to Red Copper Sites Inorg. Chem. , 45, 8012-8025

19 Tipo Zero Cobre Lancaster, K.M.; Yokoyama, K.; Richards, J.H.; Winkler, J.R.; Gray, H.B. (2009) High Potential C112D/M121X (X= M, E, H, L) P. aeurignosa Azurins. Inorg. Chem., 48, 1278-1280 Lancaster et al. (2009) Type-zero copper proteins. Nature Chemistry, 1, 711-715

The distorted tetrahedral coordination sphere of C112D/M121X (X5L,F,I) azurins features a relatively short Cu–O(G45 carbonyl) bond. a–d, The Cu(II) binding sites of C112D (a, 1.9Å, PDBID: 3FQY),C112D/M121L (b, 2.1 Å, PDBID: 3FPY),C112D/M121F (c, 1.9Å, PDBID:3FQ2), and C112D/M121I (d, 1.9 Å, PDBID: 3FQ1) azurins are displayed with Cu–heteroatom bond distances indicated in Å. O atoms are red; N atoms are blue.

20 Tipos de cobre

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Concept of Malkin and Malmström (1971) Classification of Cu sites according to UV/Vis and EPR Properties Type 2 (non-blue; mononuclear)

21 Galactose Oxidase (GalOx) -una Enzima Radical de Cobre

GalOx RCH2OH + O2 RCHO + H2O2

ABS RR

EPR EPR

22 Galactose Oxidase

PDB:1GOF

23 Cu,Zn Superoxide Dismutase .- .- + O2 + O2 + 2H → O2 + H2O2

PDB:1SPD 24 Copper Nitrite Reductase (Homotrimer) - ET and catalysis - + - NO2 + 2H + e → NO + H2O

PDB:1AS6

Copper nitrite reductase

Type 1 & Type 2 Cu

Tipos de cobre

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Concept of Malkin and Malmström (1971) Classification of Cu sites according to UV/Vis and EPR Properties Type 3 (diamagnetic ox./ed.; dinuclear)

27 Hemocyanin (O2 binding)

PDB: 1HCY, 1OXY 28 Catechol Oxidase

PDB:1BT3

29 Ascorbate Oxidase (AO), a Multi-Copper Oxidase + - 4H + 4e + O2 → 2 H2O (electrons from L-ascorbate/vitamin C Ninguna conservación de la energía !

PDB:1AOZ

30 El tipo 1 Cu y Trinuclear Cu sitio in AO

31 El sitio trinuclear reducido (izquierdo) y H2O2 (derecho) adduct de la reacción AO en crystallo

32 Caminos de Transferencia electrónica en AO

33 Respiración de Mitochondrial – Conservación de la energía Cytochrome c oxidase, una bomba de protón redox-conducida

34 Cytochrome c oxidase – Conservación de la energía La bomba de protón redox-conducida de la naturaleza

+ + - + O2 + 4H + 4Hi + 4e  H2O + H2O + 4Ho (+ 818 mV) metals (CuA, Fe-heme, Mg, Zn) e- transfer (redox; tyrosyl radical ?); H+transfer (pump)

metal centers: CuA  ET; Fe-CuB  O2 reduction

35 Repetición - Por favor, recuerde ! http://en.wikipedia.org/wiki/Copper_proteins J.T. Rubino, K.J. Franz, Journal of Inorganic Biochemistry 107 (2012) 129–143

36 Mitochondrial Cytochrome c oxidase (COX) (representation of the monomer from bovine heart/13 subunits) Tsukihara et al., SCIENCE 1995, 269, 1069; Yoshikawa et al., SCIENCE 1998, 280, 1723

Bacterial COX from Pseudomonas denitrificans (three subunits; Iwata et al., NATURE, 1995, 376, 660)

PDB:1OCC,1QLE 37 Centros metálicos en bacteriano COX Cu Fe Mg

38 Sitio de Reducción O2 (Fe(III)-Cu(II) State – covalent link Tyr-His)

39 Representación esquemática del Ciclo Catalítico (ninguna liberación de ROS tóxico; tyrosine Y radical)

40 Caminos de Transferencia de protón en bacteriano COX (D Asp 124 & E Glu 78)

41 Bioengineering de Centros de Transferencia electrónica: Bocacalle de un Tipo 1 Cu en un Dinuclear CopperA Variado-Valent Work of G Canters/Leiden/NL and Y Lu, Urbana/USA; MG Savelieff, Y Lu, J Biol Inorg Chem, 15, 967-976 (2010)

Plastocyanin/Photosynthesis Cytochrome c Oxidase/Respiration 42 Nitrous Oxide Reductase/Denitrification El Ciclo de Nitrógeno Biogeochemical

43 Reducción de N2O a N2, estrictamente proceso de dependiente de Cu

N2OR + - N2O + 2H + 2e  N2 + H2O

Eo = +1.76 V  but kinetically inert molecule; ~ 59 kcal/mol activation barrier for thermal decomposition

N N O N N O

44 N2OR de Paracoccus denitrificans (forma inactiva; purificado en la presencia de O2) Head-to-tail Homodimer - CuA ET/CuZ CAT Haltia et al., Biochem. J. (2003)

45 La valencia variada CuA Centro conservada en an/aerobes!

N2O Reductase y Cytochrome c Oxidase formal oxidation state of Cu(1.5+); 1 single electron/Cu(1+)Cu(2+)

Cu-Cu Bond ? (Metallic Cu) 46 Enganche de CO Oxidation a la Formación de Dihydrogen en Carboxydothermus hydrogenoformans Dobbek et al., SCIENCE, 2001

CO + H2O → CO2 + H2 ΔGo’ = -20 kJ/mol

+ - CO + H2O → CO2 + 2H + 2e

CODH highly complex multi-centered enzymes (Mo, FeS, FAD, Cu, Ni) found in aerobic and anaerobic microorganisms 47 A Mo-S-Cu Cluster in CODH Dobbek et al., PNAS 2002; Dobbek et al., Science 2007

CO-Dehydrogenase from Oligotropha carboxidovorans

+ - CO + H2O → CO2 + 2 H + 2e

MoCo, molybdopterin dithiolene 48 La Bioquímica de Oxidación deMetano a Metanol

Hakemian, Rosenzweig Annu. Rev. Biochem. (2007), 76:223–41 The pMMO trimer [Protein Data Bank (PDB) accession code 1YEW] with one protomer highlighted.

49 Liebermann, Rosenzweig, Nature (2005), 434, 177 Kim et al., Science (2004), 305, 1612 50 Cu/Fe Homeostasis Enfermedades Neurodegenerativas

• L. Gehrig‘s disease/SOD (McCabe et al., PNAS, 1995; Amyotrophic Lateral Sclerosis) • Fe uptake (Stearman et al., Science, 1996) • Alzheimer‘s disease (Multhaup et al., Science, 1996) • Cu transport in cells (J. Selverstone Valentine et al., Science, 1997) • Cu binding to prion proteins (D.R. Brown et al., Nature, 1997) • Cu chaperone for SOD (Rae et al., Science, 1999; Harrison et al., TIBS, 2000)

Modelo de cobre neuronal homeostasis List of abbreviations: APP, amyloid precursor protein; Atox1, Cu chaperone for ATP7A/B; ATP7A, Menkes P-type ATPase; CcO cytochrome c oxidase; CCS, copper chaperone for superoxide dismutase; Cox11, cytochrome c oxidase assembly protein; Cox17, Cu chaperone for cytochrome c oxidase; hCtr1, human Cu transporter-1; MT3, metallothionein-3; PrPc, prion protein cellular form; Sco1, cytochrome oxidase deficient homologue 1; SOD1, Cu/Zn superoxide dismutase

Que et al. (2008) Metals in Neurobiology: Probing Their Chemistry and Biology with Molecular Imaging. Chem. Rev.,108, 1517–1549.

52 Resumen y Conclusiones

Cu, un (bio)catalizador potente

Conserved ET sites Type 1 Cu & CuA (plastocyanin, NiR, COX, NOR)

Sites in enzymes Trinuclear Cu in ascorbate oxidase, Tetranuclear CuZ (sulfide) in N2OR Heterometal sites in SOD (Zn/Cu), COX (Fe/Cu), CODH (Mo/Cu)