10. ELECTRON TRANSPORT AND OXIDATIVE PHOSPHORYLATION
463
It is now apparent that each of the four oxidation steps of the cycle
can support phosphorylation. However, only one of the steps can be
clearly isolated, specifically the oxidation of «-ketoglutarate. In order to
determine the maximum P/O ratio which can be obtained, it is necessary
to confine the reaction to a single oxidation step. In the case of a-ketoglutarate, the further oxidation of succinate can be effectively inhibited
by the addition of malonate.
P/O ratios for oxidation of α-ketoglutarate have been obtained
which are significantly greater than 3.0 (146-149, 154). This means
that the number of phosphorylations which occur during oxidation of
α-ketoglutarate is 4.0. Although objections were raised in favor of the
lower value of 3.0 (155) in heart tissue, the question has now been
settled in favor of 4.0 (137, 146 148). The probable number of phosphorylations associated with the other oxidation steps of the cycle are
as follows: succinate, 2 (141, 146, 148, 154); malate, 3 (157, 141, 148);
and citrate, 3 (137, 141, 146). In addition, the oxidation of pyruvate to
acetyl CoA undoubtedly will give rise to 3 high-energy phosphate bonds
(137,141, 146, 156). Glutamate oxidation appears to proceed aerobically
with P/O ratios approaching 3.0 (141, 146, 148).
2. Oxidation of Reduced Pyridine Nucleotides
Work in Lehninger's laboratory was the first to show conclusively
that phosphorylation reactions occurred at sites along the cytochrome
respiratory chain between the pyridine nucleotides and oxygen. Experiments with radioactive orthophosphate revealed incorporation of phosphate into an acid-labile organic phosphate (157), and a P/O ratio of
1.89 (uncorrected) was observed with the aerobic oxidation of DPNH
by rat liver mitochondria (158).
Some interesting observations relating to the oxidation of external
DPNH were made in these experiments. The oxidation of DPNH did
not occur unless external cytochrome c (1.5χ 10~
4 Μ) was added; on
the other hand the oxidation of ß-hydroxybutyrate and fatty acid by
the same preparation was increased only slightly by the addition of
cytochrome c. Moreover, higher P/O ratios with DPNH as substrate
were obtained with mitochondria which had been pretreated with H 2 0
before their phosphorylating ability was measured. Such pretreated particles required much lower concentrations of cytochrome c for maximal
oxidation rates. These results are best interpreted as showing that pretreatment can greatly increase the permeability of mitochondria to external DPNH and permits access of DPNH to internal sites of phosphorylation (137, 159).
The fact that chemically reduced DPNH can support oxidative
463
It is now apparent that each of the four oxidation steps of the cycle
can support phosphorylation. However, only one of the steps can be
clearly isolated, specifically the oxidation of «-ketoglutarate. In order to
determine the maximum P/O ratio which can be obtained, it is necessary
to confine the reaction to a single oxidation step. In the case of a-ketoglutarate, the further oxidation of succinate can be effectively inhibited
by the addition of malonate.
P/O ratios for oxidation of α-ketoglutarate have been obtained
which are significantly greater than 3.0 (146-149, 154). This means
that the number of phosphorylations which occur during oxidation of
α-ketoglutarate is 4.0. Although objections were raised in favor of the
lower value of 3.0 (155) in heart tissue, the question has now been
settled in favor of 4.0 (137, 146 148). The probable number of phosphorylations associated with the other oxidation steps of the cycle are
as follows: succinate, 2 (141, 146, 148, 154); malate, 3 (157, 141, 148);
and citrate, 3 (137, 141, 146). In addition, the oxidation of pyruvate to
acetyl CoA undoubtedly will give rise to 3 high-energy phosphate bonds
(137,141, 146, 156). Glutamate oxidation appears to proceed aerobically
with P/O ratios approaching 3.0 (141, 146, 148).
2. Oxidation of Reduced Pyridine Nucleotides
Work in Lehninger's laboratory was the first to show conclusively
that phosphorylation reactions occurred at sites along the cytochrome
respiratory chain between the pyridine nucleotides and oxygen. Experiments with radioactive orthophosphate revealed incorporation of phosphate into an acid-labile organic phosphate (157), and a P/O ratio of
1.89 (uncorrected) was observed with the aerobic oxidation of DPNH
by rat liver mitochondria (158).
Some interesting observations relating to the oxidation of external
DPNH were made in these experiments. The oxidation of DPNH did
not occur unless external cytochrome c (1.5χ 10~
4 Μ) was added; on
the other hand the oxidation of ß-hydroxybutyrate and fatty acid by
the same preparation was increased only slightly by the addition of
cytochrome c. Moreover, higher P/O ratios with DPNH as substrate
were obtained with mitochondria which had been pretreated with H 2 0
before their phosphorylating ability was measured. Such pretreated particles required much lower concentrations of cytochrome c for maximal
oxidation rates. These results are best interpreted as showing that pretreatment can greatly increase the permeability of mitochondria to external DPNH and permits access of DPNH to internal sites of phosphorylation (137, 159).
The fact that chemically reduced DPNH can support oxidative