Cobre Proteínas y Enzimas - [DePa] Departamento de...
Transcript of Cobre Proteínas y Enzimas - [DePa] Departamento de...
CURSO QUÍMICA BIOINORGÁNICA
UNAM Octobre 15/17, 2012
PETER M.H. KRONECK, Lab 212
Cobre – Proteínas y Enzimas
Hans Freeman, Sidney, 1929-2008
Cuprum, from Cyprium – Metal of Cyprus
Bo G. Malmström, Göteborg,
1927-2000
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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)
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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
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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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2
4
(
mM
-1 c
m-1
)
Magnetic field (G)
Wavenumber (cm-1)
EPR
ABS
2800 3000 3200 3400
Edward I. Solomon, Stanford
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Los elementos de vida www.webelements.com
Abundancia en el cuerpo (75 kg)
Ca: 1.2 kg
K: 150 g
Na: 70 g
Mg: 20-30 g
Fe: 4-7 g
Zn: 2-3 g
Cu: 70-100 mg
Mn: 10 mg
S: 140 g
P: 780 g
H He
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
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Formas de Vida – De Anaerobio a Aerobio
condiciones anóxicas (-O2) contra condiciones óxicas (+O2)
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Cu y O2 – Una estrecha relación
ss
ss*
sp*
sp
p*
p
3S+
O2
O2-
H2O2
OH•+H2O
2H2O
-0.33V
+0.94V
+0.38V
+2.31V
E‘o vs NHE, pH 7.25
112 pm
133 pm
149 pm
1554 cm-1
1145 cm-1
842 cm-1
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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)
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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
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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)
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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
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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)
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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 ?
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Tipos de cobre
Concept of Malkin and Malmström (1971)
Classification of Cu sites according to UV/Vis
and EPR Properties
Type 1 (blue; mononuclear)
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mM
-1 c
m-1
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Magnetic field (G)
Wavenumber (cm-1)
EPR
ABS
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Plastocyanin: Blue Type 1 Cu Sitio Función: Proteína de Transferencia electrónica
(Fotosíntesis)
41% Cu 38-45% S
Cu(II) Spin-Distribution
Covalent Cu-Cys p-bond is mainly responsible
for its unique properties
EI Solomon, Inorg. Chem. 2006, 45, 8012-8025
PDB Code: 1PLC
HC Freeman, 1978 18
Edward I. Solomon (2006)
Spectroscopic Methods in Bioinorganic Chemistry: Blue to Green to Red Copper Sites
Inorg. Chem. , 45, 8012-8025
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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.
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Tipos de cobre
Concept of Malkin and Malmström (1971)
Classification of Cu sites according to UV/Vis
and EPR Properties
Type 2 (non-blue; mononuclear)
25000 20000 15000 100000
2
4
(
mM
-1 c
m-1
)
Magnetic field (G)
Wavenumber (cm-1)
EPR
ABS
2800 3000 3200 3400
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Galactose Oxidase (GalOx) -una Enzima Radical de Cobre
RCH2OH + O2 RCHO + H2O2 GalOx
RR
EPR EPR
CD
ABS
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Tipos de cobre
Concept of Malkin and Malmström (1971)
Classification of Cu sites according to UV/Vis and
EPR Properties
Type 3 (diamagnetic ox./ed.; dinuclear)
25000 20000 15000 100000
2
4
(
mM
-1 c
m-1
)
Magnetic field (G)
Wavenumber (cm-1)
EPR
ABS
2800 3000 3200 3400
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Ascorbate Oxidase (AO), a Multi-Copper Oxidase 4H+ + 4e- + O2 → 2 H2O (electrons from L-ascorbate/vitamin C
Ninguna conservación de la energía !
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PDB:1AOZ
Respiración de Mitochondrial – Conservación de la energía
Cytochrome c oxidase, una bomba de protón redox-conducida
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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
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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
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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)
37 PDB:1OCC,1QLE
Representación esquemática del Ciclo Catalítico
(ninguna liberación de ROS tóxico; tyrosine Y radical)
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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
Nitrous Oxide Reductase/Denitrification 42
Reducción de N2O a N2, estrictamente proceso de dependiente de Cu
N2O + 2H+ + 2e- N2 + H2O
Eo = +1.76 V but kinetically inert molecule; ~ 59
kcal/mol activation barrier for thermal decomposition
N2OR
N N O N N O
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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)
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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
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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
Hakemian, Rosenzweig
Annu. Rev. Biochem. (2007),
76:223–41
The pMMO trimer [Protein
Data Bank (PDB) accession code
1YEW] with
one protomer highlighted.
La Bioquímica de Oxidación
deMetano a Metanol
Liebermann, Rosenzweig, Nature (2005), 434, 177 49
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)
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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.
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