258
TABLE 14-3
Energy-Yielding Reactions in the Complete Oxidation
of Glucose
Net Moles of ATP
Generated per
Reaction
Mole of Glucose
Glycolysis
(phosphoglycerate kinase, pyruvate
kinase; two ATPs are expended)
2
NADH shuttle
glycerol-phosphate shuttle (or malate
aspartate shuttle)
4(6)
Pyruvate dehydrogenase (NADH)
6
Succinyl CoA synthetase
(GTP is equivalent to ATP)
2
Succinate dehydrogenase
(succinate —>fumarate + FADH2)
4
Other TCA cycle reactions
(isocitrate
—>
a-ketogiutarate,
0
£-ketoglutarate —> succinyl CoA,
malate—>oxaloacetate; total of 3 NADH
generated)
18
Total
36(38)
next. The energy is ultimately stored as a phosphoric
acid anhydride bond in ATP. The formulation, in general
terms, is
AH2 +
B
+ C ^ A ~
C
+ BH2
A
~
C
+ ADP + P; ^
A +
C
+
ATP
Sum:
AH2
+ B
+
ADP + P; ^ A +
BU2
+ ATP
where A and B represent the known redox pair, C is a hypo-
thetical ligand, and A ~ C is a hypothetical high-energy
intermediate. The above mechanism can be modified to
include other phosphorylated intermediates.
A model reaction that supports the above mechanism
is the glycolytic substrate-linked phosphorylation, which
proceeds via a thiol ester prior to the formation of the
phosphorylated intermediate (Chapter 13). Although the
chemical hypothesis is consistent with the substrate-linked
phosphorylation mechanism, it is deficient in explaining
the oxidative phosphorylation in mitochondria for two
reasons:
1. The postulated high-energy chemical intermediates,
either phosphorylated or nonphosphorylated, have
never been identified despite many attempts to find
them, and
2. The chemical-coupling mechanism does not explain
why the inner mitochondrial membrane must be
present as a completely closed vesicle for oxidative
phosphorylation to occur.
The
conformational hypothesis
proposes that the
energy-yielding steps generate protein conformational
changes that are used in ATP synthesis. The conforma-
tional changes that occur in the redox catalysts are trans-
mitted to the energy-transducing units via protein-protein
interactions, the formation of covalent intermediates, or
the proton-motive force. Current opinion holds that the
conformational changes are linked with a proton-motive
force (see below).
Morphological changes do occur in the inner mem-
branes of the mitochondria when active respiration is
stimulated by ADP. Fluorescent probes, such as
1-
aminonaphthalene-
8
-sulfonate (ANS) and the antibiotic
aurovertin, bind either to the inner membrane (ANS) or di-
rectly to ATP synthase (aurovertin). The binding enhances
or diminishes fluorescence in response to changes in con-
formation or hydrophobicity of the inner membrane. Re-
sults support the hypothesis that ATP synthase undergoes
conformational changes during respiration and oxidative
phosphorylation (discussed later).
According to the
chemiosmotic hypothesis,
developed
by Peter Mitchell, an electrochemical gradient (pH gradi-
ent), generated across the inner mitochondrial membrane
by the passage of reducing equivalents along the respira-
tory chain provides the driving force for the synthesis of
ATP. There are three prerequisites for achieving oxidative
phosphorylation according to this hypothesis:
1. An anisotropic (direction-oriented)
proton-translocating respiratory chain capable of
vectorial transport of protons across the membrane;
2. A coupling membrane impermeable to ions except via
specific transport systems; and
3. An anisotropic ATP synthase whose catalytic activity
is driven by an electrochemical potential.
The transport of reducing equivalents in the respiratory
chain generates a proton gradient across the membrane
by virtue of the specific vectorial arrangement of the
redox components within the inner mitochondrial mem-
brane. The proton gradient is generated by ejection of
protons from the matrix into the intermembrane space
during proton-absorbing reactions, which occur on the
M side of the inner membrane, and the proton-yielding
reactions, which occur on the C side, to form redox loops
(Figure 14-13).
According to the chemiosmotic hypothesis, ejection of
two or more protons occurs at each of three sites in com-
plexes I, III, and IV. Thus, in the transfer of two reducing
equivalents from NADH to oxygen, at least six protons
chapter 14
Electron Transport and Oxidative Phosphorylation
