Lecture 3 GLUT, NADH, TCA cycle, ETC and oxidative phosphorylation

Key takeaways

• when you eat meal, blood sugar levels increase

• insulin released so that liver stores glucose as glycogen

• blood sugar levels maintained

• if too low, glucagon released to tell liver to break glycogen into glucose

• homeostasis maintained

GLUT

Glucose Transport Proteins

GLUT1

ubiquitously distributed, red blood cells; constitutive transport

GLUT2

intestine, liver, kidney, pancreas

GLUT3

CNS, brain

GLUT4

skeletal muscle, adipose tissue, heart (insulin-regulatable)

Mechanism of glucose uptake in skeletal muscle

1. insulin binds to its receptor

2. signalling cascade releases GLUT4 vesicle

3. translocation

4. GLUT4 vesicle integrated in plasma membrane

5. facilitated glucose transport

oxidation of glucose

glucose + 6O2 = 6CO2 + 6H2O

glycolysis partial oxidation of glucose net equation

glucose + 2pi + 2ADP + 2NAD+ = 2ATP + 2NADH + 2 pyruvate + 2H+ + 2H2O

NADH is

reducing equivalent

reducing equivalents

carry electrons that can be transfered btw molecules

coenzymes that act as electron carriers

FAD, NAD+ and NADP

electron carriers are reduced by

adding electrons

NAD+ is derived from

vitamin B3

FADH2 and NADH are generated from

oxidation (ex. glycolysis, beta oxidation, TCA cycle)

anabolism

• synthesis

• reductive bc gain to build

• reducing equiv NADPH & ATP

• catalyzed by reductases

catabolism

• degradation

• oxidative bc loose to break

• makes reducing equiv NADH, FADH2, ATP

• via dehydrogenase

pyruvate is more ____ than glucose

oxidized

NAD+ is ___ form

oxidized bc lost electrons

NADH is ___ form

reduced bc has elextrons

obligate aerobes

Grow only in presence of oxygen

Facultative anaerobes

• Can grow with or without oxygen

• annelids, mollusks and some yeast

obligate anaerobes

Cannot grow in oxygen and metabolize glucose only anaerobically

lactic acid bacteria

• used for fermenting yogurt, cheese

• generate ATP by fermentation of sugars to lactate

yeast

converts pyruvate to ethanol in wine- and beer-making

after glycolysis

complete oxidation of glucose in mitochondria

mitochondria

• oxidize metabolic fuels to generate energy

• similar to power plants

• biological oxidations are catalyzed by enzymes

pyruvate is oxidized by

pyruvate dehydrogenase

Pyruvate is transported into the mitochondria via

pyruvate-H+ symport

pyruvate dehydrogenase catalyzes

irreversible oxidative decarboxylation of pyruvate to acetylCoA

Pyruvate (3C) + CoA + NAD+ =

acetylCoA (2C) + CO2 (1C) + NADH

CoA

• coenzyme A

• derivative of vitamin B

• pathogenic acid (rich in poultry, yogurt, avacado)

• acts as carrier

bond btw acetyl and CoA is

high energy thioester bond

entry of acetyl CoA into TCA completes

oxidation of glucose to CO2

TCA enzymes are

• compartmentalized

• soluble in matrix EXCEPT succinate dehydrogenase (membrane protein)

TCA equation

3NAD+ + FAD + GDP + Pi + acetylCoA = 3 NADH + FADH2 + GTP + CoA + 2CO2

TCA cycle beings and ends with

oxaloacetate

product of first reaction in TCA =

citrate = tricarboxylic acid

step 1
simps

Citrate Synthase

step 2
ask

Aconitase

step 3
dumb

Isocitrate Dehydrogenase

step 4
daughters

A-ketoglutarate Dehydrogenase

step 5
sucking

Succinyl-CoA Synthase

step 6
d*ck

Succinic Dehydrogenase

step 7
for

Fumerase

step 8
drinks

Malate Dehydrogenase

summary of TCA

• First step combines 4-C oxaloacetate with 2-C acetylCoA to form a 6-C
  compound

• First two dehydrogenase reactions are much like PDH, producing NADH
  and removing 2 CO 2 molecules

• The third dehydrogenase reaction produces FADH 2

• The final step of the cycle is the fourth dehydrogenase that produces the
  third NADH

• The first reaction is catalyzed by a synthase (no ATP involved) whereas
  the fifth reaction is catalyzed by a synthetase (involves ATP/GTP being
  used or synthesized, like a kinase)

glucose oxidation (start to finish)

• Glucose has now been completely
  oxidized to CO 2

• Electrons removed from glucose by 6 dehydrogenases were transferred to
  reducing equivalents
  • Recall that 2e - are required to reduce

• NADH and FADH 2 become reoxidized by
  passing their electrons to the ETC

mitochondria anatomy

• Outer membrane (OM) encapsulates
  the mitochondrion

• Inner membrane (IM) is invaginated,
  forming cristae

cristae

increase the surface area of
the inner membrane

inner membrane

• greater amount of
  protein than OM

• proteins of ETC,
  oxidative phosphorylation and
  transport proteins

• Number of cristae varies with
  oxidative capacity of the tissue
  (liver has very few vs muscle and heart have more)

matrix

• Contains PDH, enzymes for TCA cycle, β-
  oxidation and amino acid oxidation,
  mitochondrial DNA

• # of mitochondria varies (Exercise training increases number of
  mitochondria in muscle)
  

outer membrane is permeable via

porin

Inner membrane is relatively impermeable

• Permeable to only to small uncharged compounds O2 , CO2 , H2 O

• Contains a variety of transport proteins (translocators) for ATP, ADP, pyruvate, Pi , H+ which are all charged and very hydrophilic

*relative impermeability creates conc gradient

transport systems required for inner membrane for transport of

1. ATP
   2. ADP + Pi
   3. NADH electrons

ATP

(produced in the mitochondria) into the cytosol to be used by ATP-consuming processes

ADP + Pi

generated from ATP utilization in the cytosol) into the
mitochondria to be resynthesized into ATP by oxidative phosphorylation

NADH

(generated from glycolysis in the cytosol) into the mitochondria for ETC

ADP-ATP Translocator

• Exports ATP to cytosol in exchange for ADP (ATP out of matrix, ADP into matrix)

• Driven by membrane potential difference

• drives ATP outside of mitochondria since since ADP
  has 3 –ve charges and ATP has 4 –ve charges

P i -H + translocase

• P i (H 2 PO 4- ) generated from ATP hydrolysis is transported into the
  mitochondria via a symporter along with H +

• Driven by the electrochemical gradient of H +

• proton gradient drives H + to equilibrate, by H + entering the mitochondria
  via symport with P i (electroneutral)

Malate-aspartate shuttle: transport of NADH electrons

• cell maintains separate pools of NADH in mitochondria and cytosol

• NADH generated in the cytosol by glycolysis transfers electrons into the
  mitochondria via malate intermediate in a very complex transport
  mechanism

Malate-aspartate shuttle: transport of NADH electrons

• e - s donated from NADH to oxaloacetate to yield malate by cytosolic
  malate dehydrogenase
  2. Malate is transported into mitochondria by malate-α-ketoglutarate
  transporter
  3. Malate reoxidized to oxaloacetate, transferring e - s to NAD+ by
  mitochondrial malate dehydrogenase, yielding NADH in the
  mitochondria for ETC
  4. Oxaloacetate cannot cross the IM, \is converted to aspartate by
  mitochondrial aspartate aminotransferase (transfers amino group)
  5. Aspartate is transported into cytosol by glutamate-aspartate
  transporter
  6. Aspartate reconverted to oxaloacetate by cytosolic aspartate
  aminotransferase

malate aspartane shuttle 2 enzymes

separate mitochondrial and cytosolic pools
1.malate dehydrogenase (1 & 3)
2. aspartate aminotransferase (4 & 6)

malate aspartane shuttle 2 transporters

to exchange aspartate and α-ketoglutarate for
oxaloacetate/aspartate conversion
1.malate-α-ketoglutarate transporter (2)
2. glutamate-aspartate transporter (5)

glucose has been oxidized to co2 via

glycolysis pyruvate dehydrogenase, TCA cycle

ETC

• Composed of 4 protein complexes in inner mitochondrial
  membrane and two carrier proteins
  – Complexes I, II, III, IV
  – Coenzyme Q (Q) and Cytochrome c (Cyt c

final electron acceptor

oxygen

2 functions of protein complexes 1-4

1. shuttle electrons

2. pump protons

shuttle electrons

Through redox centres with progressively greater reduction
potential
– Proteins themselves are not reversibly reduced/oxidized
– Complexes contain combination of two or more redox centers
• Coenzymes
• Fe-S clusters
• Cytochromes
• Cu

pump protons

By harnessing the free energy released from electron transport
• Complex I – accepts electrons from NADH and pumps 4 protons
• Complex II – accepts electrons from FADH 2 (does not pump!)
• Complex III – pumps 4 protons
• Complex IV – pumps 2 protons
20

Complex I

accepts electrons from NADH and pumps 4 protons

Complex II

accepts electrons from FADH 2 (does not pump!)

Complex III

pumps 4 protons

Complex IV

pumps 2 protons

NADH uses

complex I, III, IV

FADH2 uses

complexes II, III, IV

complex II contains

TCA enzyme succinate dehydrogenase

FAD is

prosthetic group covalently bound to succinate
*As FADH 2 is formed by succinate dehydrogenase it is readily
reoxidized by Complex II

complexes I, III, IV uses

free energy of electron transport to pump H+ from matrix to intermembrane space
this generates electrochem gradient across inner memb

oxidative phosphrylation

• potential energy in the electrochemical gradient of protons is coupled to
  oxidative phosphorylation of ATP

• free energy released upon re-entry of protons into the matrix is
  harnessed by ATP synthase to drive the phosphorylation of ADP

peter d mitchhell nobel prize winner

ubiquinone and proton pump

F 1 F 0 -ATPase

made up of two functional domains held together by a protein stalk
F1 + protein stalk + F0

F1

contains ATP synthetase enzyme

protein stalk

binds F1 to F0

F0

proton channel that spans IM

complexes I and III pump

4H+

complex IV pumps

2H+

complex II

electron transfer does not provide enough energy to pump protons

NADH pumps

10 protons
yielding 3 ATP ratio

FADH2

pumps 6 protons
yeilding 2 ATP

partial oxidation of glucose through glycolysis yields

2 ATP

complete oxidation of glucose yeilds

38 ATP