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 ABS 4 ) 1 - m c 1 - M m 2 ( 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 ss 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 ? 16 Tipos de cobre Concept of Malkin and Malmström (1971) ABS 4 ) 1 - m c 1 - M m 2 ( Classification of Cu sites according to UV/Vis 0 25000 20000 15000 10000 and EPR Properties Wavenumber (cm-1) EPR 2800 3000 3200 3400 Type 1 (blue; mononuclear)Magneti c field (G) 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 Concept of Malkin and Malmström (1971) ABS 4 ) 1 - m c 1 - M m 2 ( Classification of Cu sites according to UV/Vis 0 25000 20000 15000 10000 and EPR Properties Wavenumber (cm-1) EPR 2800 3000 3200 3400 Type 2 (non-blue; mononuclear)Magnetic field (G) 21 Galactose Oxidase (GalOx) -una Enzima Radical de Cobre GalOx RCH2OH + O2 RCHO + H2O2 ABS RR CD 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 25 Copper nitrite reductase Type 1 & Type 2 Cu 26 Tipos de cobre Concept of Malkin and Malmström (1971) ABS 4 ) 1 - m c 1 - M m 2 ( Classification of Cu sites according to UV/Vis and 0 25000 20000 15000 10000 EPR Properties Wavenumber (cm-1) EPR 2800 3000 3200 3400 Type 3 (diamagnetic ox./ed.;Magn etic fidinuclear)eld (G) 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.