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

1

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

25000 20000 15000 100000

2

4

(

mM

-1 c

m-1

)

Magnetic field (G)

Wavenumber (cm-1)

EPR

ABS

2800 3000 3200 3400

Edward I. Solomon, Stanford

5

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

6

Formas de Vida – De Anaerobio a Aerobio

condiciones anóxicas (-O2) contra condiciones óxicas (+O2)

7

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

8

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

His

Lys

Tyr

Glu(+Asp)

Ser

Cys

Met

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)

Classification of Cu sites according to UV/Vis

and EPR Properties

Type 1 (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

17

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

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)

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

21

Galactose Oxidase (GalOx) -una Enzima Radical de Cobre

RCH2OH + O2 RCHO + H2O2 GalOx

RR

EPR EPR

CD

ABS

22

Galactose Oxidase

23

PDB:1GOF

Cu,Zn Superoxide Dismutase

O2.- + O2

.- + 2H+ → O2 + H2O2

24 PDB:1SPD

Copper Nitrite Reductase (Homotrimer) - ET and catalysis

NO2- + 2H+ + e- → NO + H2O

25

PDB:1AS6

Copper nitrite reductase

Type 1 & Type 2 Cu

26

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

27

Hemocyanin (O2 binding)

28 PDB: 1HCY, 1OXY

Catechol Oxidase

29

PDB:1BT3

Ascorbate Oxidase (AO), a Multi-Copper Oxidase 4H+ + 4e- + O2 → 2 H2O (electrons from L-ascorbate/vitamin C

Ninguna conservación de la energía !

30

PDB:1AOZ

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)

37 PDB:1OCC,1QLE

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

Nitrous Oxide Reductase/Denitrification 42

El Ciclo de Nitrógeno Biogeochemical

43

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

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

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

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)

51

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)

53