1. OXIDATION PHOSPHORYLATION-2
BIOC DR. TISCHLER
LECTURE 29
OXIDATION PHOSPHORYLATION-2

2. OBJECTIVES
How pH and charge gradients form across mitochondrial inner membrane
Electroneutral vs electrogenic transport; significance of electrogenic transport for adenine nucleotide transporter; role of this transporter.
Chemiosmotic model.
Components of the ATP synthase complex; describe their roles.
How the malate-aspartate and -glycerol phosphate electron shuttles generate energy from NADH produced by glycolysis.
Define respiratory control and uncoupling; physiological importance of these processes.

3. + + + + + + + Outer Membrane - Inner Membrane - - - - - -
High [H+]
High [H+] + + + + + + H+
Outer
Membrane - -
Inner
Membrane - - - - - - - _ - _
Low [H+]
Intermembrane
Space +
Matrix H+ + + H+ + + +
Cytoplasm + + + + + H+ H+
Figure 1. Generation of a pH gradient ([H+]) and charge difference (negative in the matrix) across the inner membrane constitute the protonmotive force that can be used to drive ATP synthesis and transport processes

4. – MITOCHONDRIAL TRANSPORT SYSTEMS
Examples of Electroneutral Transport:
Pyruvate1- moves into matrix and OH1- moves out
Phosphate1- moves into matrix and OH1- moves out
Citrate3- + H+ exchanges with malate2-
Inner
Membrane
Matrix
Side
Intermembrane
Space
ATP4-
Adenine nucleotide
transporter
ADP3-
Net negative
charge
moves out –
Electrogenic Transport
Figure 2. Electrogenic transport system in mitochondria.

5. OH-
ADP3-
ATP4-
INTERMEMBRANE SPACE
ADP3-
4H+
ATP4-
OH-
ADP3-
ADP3-
NADH
+ H+
complex I
FMNH2
NAD+
ATP4-
4H+ e-
OH-
ADP3- e-
ADP3- e-
inner
membrane e-
3H+
ATP4-
Pi-
ADP3-
ADP3-
CoQ
complex III
4H+
2H+ e-
cyt b e-
2H+ F1 C1
½O2
H2O e-
cyt
MATRIX e- C e-
a-a3
stalk e-
4H+ Fo
complex IV
Proton gradient/
Charge gradient
3H+
Figure 3. Oxidative Phosphorylation

6. FEATURES OF THE CHEMIOSMOTIC MODEL:
mitochondrial inner membrane is impermeable to protons by simple diffusion
measurable proton (pH) gradient exists across the inner membrane
collapse of the proton gradient (uncoupling) abolishes ATP synthesis but accelerates O2 consumption
inhibition of the respiratory chain prevents ATP synthesis because pumping of protons ceases

7. Figure 4. The malate-aspartate shuttle.
e = electrons
INNER
MEMBRANE
NAD+
Glucose
Pyruvate
GLYCOLYSIS
NADH
OUTER
MEMBRANE e-
Complex I e-
OAA
NADH
NAD+
(3) e-
Glu0
Asp-1
(4)
OAA
Malate
(1) e-
NAD+
Glu0
(6)
(5)
Asp-1 KG
Malate
(2) e-
CYTOPLASM
MATRIX
Figure 4. The malate-aspartate shuttle.

8. e = electrons
CoQ e O2
INNER
MEMBRANE
OUTER
MEMBRANE
CYTOPLASM
NADH
Glucose
Pyruvate
GLYCOLYSIS
NAD+
MATRIX
FADH2 e
Dihydroxyacetone
phosphate
(DHAP)
DHAP
NAD+
3-phosphate
Glycerol e
(1)
(2)
Glycerol-3-phosphate
dehydrogenase
G3P
FAD
Figure 5. Glycerol phosphate shuttle. Cytoplasmic glycerol 3-phosphate dehydrogenase (1) oxidizes NADH. Glycerol 3-phosphate dehydrogenase in the inner membrane (2) reduces FAD to FADH2.

9. RESPIRATORY CONTROL
depends on the availability of ADP
increased ADP in matrix opens proton channel
protons move through channel down pH gradient
respiration increases to compensate for decline in pH gradient; oxygen consumption (respiration) controlled
at low ADP, ATP synthesis ceases, pH gradient builds up, oxygen use diminishes
as ATP needs rise (i.e., ADP increases) respiration is again accelerated
inhibition of respiratory chain causes loss of control

10. UNCOUPLER EFFECTS
hydrophobic molecules that bind protons
take protons into matrix to collapse pH gradient
without pH gradient, synthesis of ATP ceases
electron transport chain operates at high rate; protons are pumped out rapidly in attempt to restore pH gradient
energy is released as heat and the body temperature rises
respiratory control is lost
uncoupled brown fat mitochondria generates body heat in infants until shivering reflex develops