metabolic biochemistry lecture 8 aug. 23, 2006 oxidative phosphorylation
TRANSCRIPT
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Metabolic Biochemistry
Lecture 8 Aug. 23, 2006
Oxidative Phosphorylation
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Wild type SDHC
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Outer membraneInner membrane
Intermembrane spaceMatrix
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LNC Fig.19.7
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the arrangement of the complexes in the inner membrane
and
the order of electron flow
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In the presence of an inhibitor, all the complexes upstream of the block are reduced (blue), and all the complexes downstreamof the block are oxidized: these determinations can be made by spectroscopic measurements with isolated mitochondria.
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NADH
MEASUREMENTS WITH AN OXYGEN ELECTRODE
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LNC Fig.19.15
TMPD/ascorbate
CN-
rotenone
malonate
antimycin
Glutamate/malate
succinate
Succinate dehydrogenasemembrane bound enzyme of
Krebs cycle
Four integral membrane protein complexesTwo mobile carriers: ubiquinone and cytochrome c
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LNC Fig.19.8
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oxidized
Coenzyme Q or Q
reduced
Coenzyme Q or QH2
LNC Fig.19.2
lipid-soluble polyisoprene chain
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Heme + apoprotein cytochrome
LNC Fig.19.3
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Structure of
cytochrome c
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LNC Fig.19.4
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NON-HEME IRON - SULFUR CENTERS
[Fe2-S2]
[Fe4 - S4]
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LNC Fig.19.5
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[Fe2-S2]LNC Fig.19.5
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[Fe4-S4]
LNC Fig.19.5
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LNC Fig.19.5
A bacterial ferredoxin
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NADHO2
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LNC Fig.19.9
7 or 8
NADH + Q + H+ NAD+ + QH2
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LNC Fig.19.10
Architecture of SuccinateDehydrogenase and ReactiveOxygen Species GenerationVictoria Yankovskaya,1* Rob Horsefield,2* Susanna To¨rnroth,3*Ce´sar Luna-Chavez,1,4† Hideto Miyoshi,5 Christophe Le´ger,6‡Bernadette Byrne,2 Gary Cecchini,1,4§ So Iwata2,3,7§
SCIENCE 31 JANUARY 2003 VOL 299, p.700 www.sciencemag.org
[2Fe-2S][3Fe-4S][4Fe-4S]
succinate
Succinate + Q fumarate + QH2
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Complex III8-11 polypeptidestwo cytochromes, b and c1
one iron-sulfur center
LNC Fig.19-11
QH2 + 2 cyt c (Fe+3) Q + 2 cyt c (Fe+2)(ignore the protons for the moment)
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LNC Fig.19-11b
Complex III8-11 polypeptidestwo cytochromes, b and c1
one iron-sulfur center
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LNC Fig.19-12 The Q Cycle
Net equation
QH2 + 2 cyt cox + 2H+in Q + 2 cyt cred + 4H+
out 2 Fe+3 2 Fe+2
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From AY Mulkidjanian BBA 1709: 5-34 (2005)
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Cyt c1 red + Cyt c ox Cyt c1 ox + Cyt c red
Fe+3Fe+3 Fe+2Fe+2
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LNC Fig19-13a
Complex IV - cytochrome oxidase
9 - 13 polypeptides
cytochromes a and a3
two copper centers
4 cyt c (Fe+2) + O2 + 4H+ 4 cyt c (Fe+3) + 2 H2O( and protons pumped)
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Complex IV (schematic)
LNC Fig.19-14
4 cyt cred + O2 + 4 H+
4 cyt cox + 2 H2O
Fe+2
Fe+3
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LNC Fig.19.15
Succinate + FAD fumarate + FADH2
FAD
FMN
H+ H+H+
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Proton pumping
and
Storage of Free Energy
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+++
+++
---
---
MatrixIMS
inner membrane
LNC 19-6
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G = 2.3 RT pH + 1 x F x
pH = ~ 0.75
~ 0.15 - 0.2 v = 200 mV
G = ~ +20 kJ/mol (H+)
The oxidation of NADH liberates ~ 220 kJ/mol (NADH)
therefore,
we can pump ~ 11 protons at 100% efficiency
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tightly coupled vs
uncoupled mitochondria
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succinate
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LNC 19-18a
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Effect of antibiotics valinomycin and nigericin
Valinomycin is a K+ ionophoreit breaks down the membrane potential
Nigericin is a H + /K + antiporterit exchanges protons for potassium ions and thus converts a proton gradient into a K + gradient
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ATP Synthase
Complex V
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FoF1 ATP SYNTHASE
F1:
Fo: a b2 c9-12
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The engine can turn in both directions:with ATP hydrolysis it turns in the opposite directionwhen compared with ATP synthesis driven by proton flux into the matrix
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Interesting questions:
How many protons enter per ATP produced (per 120o turn)?
How many c-subunits per subunit?
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A simple estimate
10 PROTONS pumped per NAD+ oxidized10 PROTONS pumped per OXYGEN consumed
3 PROTONS pass through the ATP synthase per 120o turn 1 ATP is made per 120o turn
Therefore: 3ATP per 360o turn
3 ATP / 9 PROTONS
3 ATP per OXYGEN (or per NADH oxidized):
P/O ratio = 3
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Experimental measurements of P/O ratios:
P/O = ~ 2.5
Is that a problem?
NO! There are other ways for protons to go back to the matrix
without passing through the ATPsynthase:COUPLING is not perfect
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Thermoregulation
Thermogenesis
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(limited amount)
In the presence of an uncoupler the P/O ratio is reduced to zero
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LNC 19-17b
Oligomycin is a highly specific inhibitor of the ATP synthase
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Uncoupling protein, UCP
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End of Lecture 8
August 23, 2006