Metabolic Biochemistry

Lecture 8 Aug. 23, 2006

Oxidative Phosphorylation

Wild type SDHC

Outer membraneInner membrane

Intermembrane spaceMatrix

LNC Fig.19.7

the arrangement of the complexes in the inner membrane

and

the order of electron flow

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.

NADH

MEASUREMENTS WITH AN OXYGEN ELECTRODE

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

LNC Fig.19.8

oxidized

Coenzyme Q or Q

reduced

Coenzyme Q or QH2

LNC Fig.19.2

lipid-soluble polyisoprene chain

Heme + apoprotein cytochrome

LNC Fig.19.3

Structure of

cytochrome c

LNC Fig.19.4

NON-HEME IRON - SULFUR CENTERS

[Fe2-S2]

[Fe4 - S4]

LNC Fig.19.5

[Fe2-S2]LNC Fig.19.5

[Fe4-S4]

LNC Fig.19.5

LNC Fig.19.5

A bacterial ferredoxin

NADHO2

LNC Fig.19.9

7 or 8

NADH + Q + H+ NAD+ + QH2

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

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)

LNC Fig.19-11b

Complex III8-11 polypeptidestwo cytochromes, b and c1

one iron-sulfur center

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

From AY Mulkidjanian BBA 1709: 5-34 (2005)

Cyt c1 red + Cyt c ox Cyt c1 ox + Cyt c red

Fe+3Fe+3 Fe+2Fe+2

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)

Complex IV (schematic)

LNC Fig.19-14

4 cyt cred + O2 + 4 H+

4 cyt cox + 2 H2O

Fe+2

Fe+3

LNC Fig.19.15

Succinate + FAD fumarate + FADH2

FAD

FMN

H+ H+H+

Proton pumping

and

Storage of Free Energy

+++

+++

---

---

MatrixIMS

inner membrane

LNC 19-6

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

tightly coupled vs

uncoupled mitochondria

succinate

LNC 19-18a

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

ATP Synthase

Complex V

FoF1 ATP SYNTHASE

F1:

Fo: a b2 c9-12

C:\Documents and Settings\Immo E. Scheffler\Desktop\BIBC102.SU06\ATPsyn.gif

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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

Interesting questions:

How many protons enter per ATP produced (per 120o turn)?

How many c-subunits per subunit?

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

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

Thermoregulation

Thermogenesis

(limited amount)

In the presence of an uncoupler the P/O ratio is reduced to zero

LNC 19-17b

Oligomycin is a highly specific inhibitor of the ATP synthase

Uncoupling protein, UCP

End of Lecture 8

August 23, 2006