Cadena de Tranasporte de elect y Fosf OX

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Dr. Juan Pablo Damián
Bioquímica
Facultad de Veterinaria
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proteínas
glucógeno
triacilglicéridos
aminoácidos
glucosa
ácidos grasos
Respiración celular
1) Producción AcetilCoA
ee-
2) Oxidación AcetilCoA
Acetil CoA
ee-
Ciclo de Krebs
CO2
e-
e3) Transferencia electrónica
Fosforilación oxidativa
e-
NADH + H+
FADH2
NAD+
FAD
e-
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CO2
ATP
ADP + Pi
Anatomía bioquímica de la mitocondria
Membrana interna
Membrana
externa
Impermeabilidad
↓
característica esencial para
la generación de energía
Permeable a Mr<5000
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Es en la mitocondria donde se realiza la
transferencia de electrones y la fosforilación
oxidativa
Membrana externa
Membrana externa
Cara externa
Citosol
Membrana interna
Cara interna
Crestas
Cara externa
Membrana interna
Cara interna
Matriz
Matriz
Complejos de la Cadena respiratoria
y ATP Sintasa
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Aceptores de electrones universales
• sustrato reducido + NAD+
 sustrato oxidao + NADH + H+
• sustrato reducido + NADP+ 
• sustrato reducido + FAD 
sustrato oxidado + NADPH + H+
sustrato oxidado + FADH2
• sustrato reducido + FMN  sustrato oxidado + FMNH2
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Tipos de transferencias electrónicas en la fosforilación oxidativa:
1.
2.
3.
Transferencia directa de e-: Fe3+ Fe2+
Transferencia de un átomo de hidrógeno (H+ + e-)
Transferencia de un ión hidruro (:H-) portador de 2 e-.
La tendencia a aceptar o ceder e- dependerá de afinidad relativa por
los e- de cada par redox (E, Potencial de reducción estándar)
Grupos transportadores de electrones:
1.
2.
3.
4.
5.
NAD
Flavoproteínas
Ubiquinona (Coenzima Q)
Citocromos
Proteínas ferro-sulfuradas
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Tipos de transferencias electrónicas en la fosforilación oxidativa:
1.
2.
3.
Transferencia directa de e-: Fe3+ Fe2+
Transferencia de un átomo de hidrógeno (H+ + e-)
Transferencia de un ión hidruro (:H-) portador de 2 e-.
La tendencia a aceptar o ceder e- dependerá de afinidad relativa por
los e- de cada par redox (E, Potencial de reducción estándar)
Grupos transportadores de electrones:
1.
2.
3.
4.
5.
NAD
Con centros activos con:
Flavoproteínas
FAD o FMN
Ubiquinona (Coenzima Q)
Citocromos
Proteínas ferro-sulfuradas
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Grupos transportadores
3) Ubiquinona
Benzoquinona liposoluble
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Grupos transportadores….
4) Citocromos a, b y c
La mayoría son proteínas transmembrana.
Citocromo c, proteína soluble asociada con
cara externa membrana mitocondrial interna
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Grupos transportadores….
4) Citocromos a, b y c
La mayoría son proteínas transmembrana.
Citocromo c, proteína soluble asociada con
cara externa membrana mitocondrial interna
Potencial de Reducción estándar del átomo de
Fe depende de la proteína
5) Proteínas ferro-sulfuradas
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Grupos transportadores….
Potencial de Reducción estándar del átomo de
Fe depende de la proteína
5) Proteínas ferro-sulfuradas
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Potenciales de reducción estándar
Medida de la afinidad por los electrones
Flujo espontáneo de e-
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Transportadores de e- están organizados
en complejos multienzimáticos
Complejo enzimático
kDa
(subunidades)
Grupo
prostético
850 (42)
FMN, Fe-S
II Succinato deshidrogenasa
140 (5)
FAD, Fe-S
III Ubiquinona: cit. c
oxidoreductasa
citocromo c
250 (11)
Hemos, Fe-S
IV Citocromo oxidasa
160 (13)
I NADH deshidrogenasa
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13 (1)
hemo
Hemos,
CuA,CuB
Complejo I: NADH-Ubiquinona oxidorreductasa
NADH + H+ + Q  NAD+ + QH2
1H+
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Complejo I: NADH-Ubiquinona oxidorreductasa
NADH + H+ + Q  NAD+ + QH2
1H+
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Complejo I: NADH-Ubiquinona oxidorreductasa
NADH + H+ + Q  NAD+ + QH2
1H+
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Complejo I: NADH-Ubiquinona oxidorreductasa
NADH + H+ + Q  NAD+ + QH2
Complejo Transmembrana
Bomba de Protones
1H+
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Complejo I: NADH-Ubiquinona oxidorreductasa
NADH + H+ + Q  NAD+ + QH2
Positivamente
Complejo Transmembrana
Bomba de Protones
Negativamente
1H+
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Complejo II: Succinato Deshidrogenasa
O
O
Succinato
C
CH2
CH2
C
O-
O
C
O
QH2
O
O
Fumarato
FAD
CH
CH
C
O-
FADH2
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Q
Complejo II: Succinato Deshidrogenasa
Ciclo de Krebs
O
O
Succinato
C
CH2
CH2
C
O-
O
C
O
QH2
O
O
Fumarato
FAD
CH
CH
C
O-
FADH2
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Q
Complejo II: Succinato Deshidrogenasa
Succinato
Fumarato
Ciclo de Krebs
O
O
Succinato
C
CH2
CH2
C
O-
O
C
O
QH2
O
O
Fumarato
FAD
CH
CH
C
O-
FADH2
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Q
Otras Deshidrogenasas
Flavoproteína transferidora de e-
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Complejo III: Ubiquinona-citocromo oxidorreductasa
Ciclo Q
QH2 + Cyt c1 (oxidado)  Q˹- + 2HP+ + Cyt c1 (reducido)
QH2 + Q˹- + 2HN+ + Cyt c1 (oxidado)  QH2 + 2HP+ + Q + Cyt c1 (reducido)
QH2 + 2 Cyt c1 (oxidado) + 2HN+  Q + 2 Cyt c1 (reducido) + 4HP+
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Complejo IV: Citocromo oxidasa
2 Cyt c (reducido) + 4HN + +
1/2O2
 2 Cyt c (oxidado) + 2Hp+ + 1H2O
4 Cyt c (reducido) + 8HN + + O2  4 Cyt c (oxidado) + 4Hp+ + 2H2O
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Mecanismos propuestos para el transporte de H+
1. Mecanismo del rodeo redox
2. Mecanismo del bombeo de H+
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Resumen del flujo de electrones y protones
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Como se determinó la secuencia de
transporte de electrones?
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1) Medición experimental de los potenciales de reducción estándar
Flujo espontáneo de e-
2) Reducción de la cadena de transporte de e- aportando una fuente de
e- en ausencia del aceptor de e- (O2)
Al introducir O2 se estudia la velocidad de oxidación de cada transportador: el
primero en ceder sus electrones es el más próximo al O2.
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3) Utilización de agentes inhibidores específicos de transportadores
DCIP
Azida de Sodio
0.7 0.8
0.6 0.7
Absorbancia (600 nm )
A bs orbanc ia (600 nm )
DCIP
Succinato
Succinato + DCIP
0.5 0.6
0.4
0.3
0.5
0.4
0.3
0.2 0.2
0.1 0.1
0
0
Extracto mitocondrias
Succinato + DCIP
Succinato + Azida + DCIP
0.8
AzulIncoloro
0
0
2
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2
4 4
6 6
88
Tiempo (minutos)
Tiempo (minutos)
10
10
Síntesis de ATP
Transferencia electrónica
La hipótesis quimiosmótica
Fuerza protón-motriz
(200 kJ/”mol” de par e-)
Síntesis de ATP
(50 kJ/mol)
Es termodinámicamente posible, pero Cómo?
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Peter Mitchell (1920-1992)
Modelo quimiosmótico
Peter Mitchell
ÄG =40-50 kJ/mol
ÄpH=0.75
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El modelo quimiosmótico (1961)
Energía libre transporte de e-  bombeo de H+ 
gradiente electroquímico de H+ síntesis de ATP
Peter Mitchell (1920-1992)
Observaciones experimentales:
1.
2.
3.
4.
La fosforilación oxidativa requiere una membrana interna intacta
La membrana interna es impermeable a iones como H+, OH-, K+, ClEl transporte de e- resulta en transporte de H+,fuera de la mitocondria
Compuestos que aumentan la permeabilidad de la membrana mitocondrial
interna disipan el gradiente electroquímico, el transporte de e- es posible pero
NO hay síntesis de ATP.
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Acoplamiento de procesos
Experiencia 1
CN-
Experiencia 1
La síntesis de ATP depende de la cadena de transporte de epdfMachine
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Acoplamiento de procesos
Experiencia 2
Oligomicina
Experiencia 2
El funcionamiento de la cadena de transporte de e- depende de la
síntesis de ATP
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Acoplamiento de procesos
Experiencia 2
Oligomicina
Experiencia 2
El funcionamiento de la cadena de transporte de e- depende de la
síntesis de ATP
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Fosforilación oxidativa
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ATP SINTASA
Matriz mitocondrial
F1
â contiene el sitio de ATP Sintasa
 es la puerta al canal de H+ (Fo es el canal)
Tallo
Une F1 con Fo
Membrana mitocondrial interna
Fo
contiene 6-10 copias de un lipoproteína (canal
de H+)
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ATP SINTASA
ADP + Pi
ATP + H2O G´= 0 kJ/mol
en superficie de la enzima
âATP
ATP + H2O
âADP
âvacía
ADP + Pi G´= - 30.5 kJ/mol
ATP libre en solución
FoF1 une ATP con alta afinidad Kd  10-12 M
FoF1 une ADP con baja afinidad Kd  10-5 M
matriz
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F1
Estado L (loose)
Estado O (open)
Estado T (tight)
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fluorescente
Demostración experimental
de la rotación de la ATP
sintasa (Yoshida & Kinosita)
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Estequiometría consumo de O2 y síntesis de ATP
xADP + xPi + ½O2 + NADH  xATP + H20 +NAD+
x: razón P/O (ATP/1/2O2)
para el NADH= 3 ATP  2.5
para el FADH2= 2 ATP  1.5
Pero no es necesario que P/O sea un número entero
Valores consenso
Cuantos H+ se bombean hacia afuera a partir del NADH? 10
Cuantos H+ se bombean hacia afuera a partir del FADH2? 6
Cuantos H+ deben entrar para sintetizar un ATP?
4
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Fuerza protón-motriz suministra energía para transporte activo
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Fuerza protón-motriz suministra energía para transporte activo
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Control respiratorio
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Fosforilación oxidativa se regula por la disponibilidad del sustrato
Mitocondrias hígado de rata
Estado: Alta energía
Estado: en reposo
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Regulación de la Fosforilación Oxidativa
Velocidad de consumo de O2  disponibilidad de ADP
Estatus energético:
[ATP ]
[ADP] [Pi]
La velocidad de oxidación de combustibles es tan
rápida que [ATP]/ [ADP] varía muy poco
Regulación de vías productoras de ATP
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Síntesis
OBJETIVOS DE LA CLASE
- Conocer los componentes de la cadena de transporte de
e- y como funcionan.
- Comprender como la energía del flujo de e- a través de
la cadena se acopla al transporte de H+ a través de
la membrana mitocondrial.
- Analizar como el flujo de H+ a favor de su gradiente de
concentración se acopla a la síntesis del ATP.
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Bibliografía
1.
2.
3.
4.
5.
Principios de Bioquímica. Lehninger, Nelson & Cox Ediciones
Omega 3ra Edición, 2001.
Biochemistry. Voet & Voet. Wiley & Sons Inc., 2da Edición, 1995.
Textbook of Biochemistry with clinical correlations. Devlin TM.
Wiley & Sons Inc., 3ra Edición, 1992.
Bioquímica. Stryer. Editorial Reverté 4ta Edición, 1995.
Biología Molecular de la Célula. Alberts y col. Ediciones Omega,
3ra Edición, 1996.
Páginas web de interés
1.
2.
3.
http://www.people.virginia.edu/
http://www.worthpublishers.com.lehninger/
http://bioquimica.online.pt
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Micrografías electrónicas
Mitocondria intacta
Partículas submitocondriales
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Partículas submitocondriales
luego de tratamiento c/urea
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Si la finalidad de la cadena es bombear
protones hacia fuera para crear el
potencial electroquímico para la síntesis de
ATP  un gradiente de protones
artificialmente
creado
debería
ser
suficiente para esta síntesis
pdfMachine
Is a pdf writer that produces quality PDF files with ease!
Produce quality PDF files in seconds and preserve the integrity of your original documents. Compatible across
nearly all Windows platforms, if you can print from a windows application you can use pdfMachine.
Get yours now!
pdfMachine
Is a pdf writer that produces quality PDF files with ease!
Produce quality PDF files in seconds and preserve the integrity of your original documents. Compatible across
nearly all Windows platforms, if you can print from a windows application you can use pdfMachine.
Get yours now!
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pdfMachine
Is a pdf writer that produces quality PDF files with ease!
Produce quality PDF files in seconds and preserve the integrity of your original documents. Compatible across
nearly all Windows platforms, if you can print from a windows application you can use pdfMachine.
Get yours now!
pdfMachine
Is a pdf writer that produces quality PDF files with ease!
Produce quality PDF files in seconds and preserve the integrity of your original documents. Compatible across
nearly all Windows platforms, if you can print from a windows application you can use pdfMachine.
Get yours now!
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