• Review of e transport and H + pumps.
• Structure and function of ATP synthase.
• Quantitation of Chemiosmotic Potential.
• ADP regulation of respiratory metabolism.
• Chemical/morphologic events of respiration.
•
Metabolite pumps of inner mitochondrial membrane.
• The Ca ++ pump, a key mitochondrial function.
•
Spectral properties of heme proteins.
• Distinguish between Oxidases and Oxygenases
•
The role of Oxygen free radicals in tissues and mechanisms that protect against tissue damage
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Key issues revisited :
1. e transport results in: a. increased transmembrane
D
[H + ]. Hi H + outside.
b. increased transmembrane
D
V (+ outside).
2. Transformation of H + energy into ATP energy requires: a. Intact membrane b. Transmembrane proton driven ATP synthase c. ADP and inorganic phosphate
Walt Balcavage 4/10/2020
Complex
I
2

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1. Free energy available from a concentration difference across a membrane :
2. In the special case where the concentration difference is due that of H+:
•
The 1st term is also known as the proton motive force (pmf).
• In the 2nd term
 is transmembrane potential in Volts.
•
The 3rd term represents difference in [
D
H + ] across the membrane
• Any combination of values for membrane potential and pH difference that sum to more than the free energy of synthesizing ATP will lead to ATP synthesis.
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1. Normally the rate of electron transport is set by the available ADP, i.e., the energy charge of the cell regulates the process.
2. When ADP is limiting: a. Flow thru ATPase slows.
b. H + accumulates in cytosol.
c. proton pumps become limited by hi pmf.
d. electron transport slows because pumps are slowed e. the rate of O
2 utilization is minimized and reduced substrates are conserved .
E.
T.
S.
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Regulation of respiration by presence/absence of ADP state 4 (resting respiration)
Electron micrographs illustrating morphological changes mitochondria undergo as they pass from resting state (top, orthodox form) to active state
( bottom . condensed form) state 3
(active respiration)
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Matrix Side
Cytosolic Side
Antiport
Symport
Antiport
Antiport
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Calcium Transport, a preeminent regulator of cell metabolism
• The mitochondrial calcium carrier is one of the most active carriers of the mitochondrial inner membrane.
• Calcium transport is driven by the pmf generated by electron transport or ATP hydrolysis.
• The mitochondrial calcium carrier is an important reservoir of cellular calcium, sequestering and releasing
Ca ++ in response to a variety of signals, e.g., depolarizing voltage in muscle cells, IP hormone sensitive cells.
3 in
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Pure Cytochrome c
Mitochondrial suspension:
(reduced - oxidized)
As apparent from the above, the reduced form (ferrous) of cyt. c is more absorbing than is the oxidized form.
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Similar spectral effects are observed when mitochondria are made reduced.
All the cytochromes are more absorbing than cytochromes in aerobic or oxidized mitochondria.
9

Oxidase
2SH2 + O 2 2S (ox) + 2H2 O
2SH2 + R + O 2
Monooxygenase
2S (ox) + H2 O + R O H
2SH2 + 2R ( R R ) + O 2
Dioxygenase
2S (ox) + 2R OH ( R O H R O H)
• Oxidases, (cyt. a+a
3 into water oxidase) incorporate molecular oxygen only
• Dioxygenases incorporate oxygen only into organic molecule,
(inter or intra molecular)
• Monooxygenase incorporate one atom of oxygen from O
2 into water and one atom of oxygen into an organic molecule. Because of this dual use of oxygen these oxygenases are often called
“mixed function oxygenases”. These enzymes are highly inducible, they act to detoxify and make soluble many otherwise deleterious compounds and they all contain the heme protein known as cytochrome P450.
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Walt Balcavage
P450
RO H R-H
Fe
+3
P450
R-H P450
RO H
Fe
+3
Fe
+3 e-
P450
MONOOXYGENASE
P450
P450
Fe O
+3
R-H
Fe
+2
R-H
H
2
O
O
2
H
+ P450
Fe O:O H
R-H
P450
Fe
+2
O:O
R-H
H
+
4/10/2020 e-
11

Tissue generation and disposition of oxygen free radicals
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Glutathione, a key subcellular reductant
Vitamin E, a dietary lipophylic antioxidant
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