10. ELECTRON TRANSPORT AND OXIDATIVE PHOSPHORYLATION
465
Other workers have demonstrated that the oxidation of isocitrate by
a TPN
+ -linked dehydrogenase in rat liver mitochondria proceeds with a
P/O ratio of approximately 1.0 (163). The evidence indicates that the
single phosphorylation step occurs when ferrocytochrome c, reduced in
the presence of TPNH and cytochrome c reductase, is oxidized by molecular 0 2 . This mechanism provides a means for obtaining some of the
potential energy available in TPNH, but the process is much less efficient
energetically than the process in which DPN
+ is reduced through the
enzyme transhydrogenase.
3. Oxidation of ß-Hydroxybutyrate
Before highly purified DPNH was available, Lehninger's group utilized the oxidation of ß-hydroxybutyrate to obtain information concerning the phosphorylation processes coupled to pyridine nucleotide oxidations (159). The oxidation of ß-hydroxybutyrate is a single step oxidation catalyzed by a DPN
+ -requiring dehydrogenase. One of the products
formed, acetoacetate, was metabolically inert in their system. The DPNH
formed was reoxidized by the respiratory chain. Various factors
(Mg
2+ , arsenite, dinitrophenol) which were known to influence oxidative
phosphorylation with DPNH also influenced phosphorylation associated
with /?-hydroxybutyrate to the same extent. There is no evidence to indicate that phosphorylation might occur at the substrate level between
substrate and DPN
+ (145). P/O ratios in excess of 2.0 have been
obtained in the oxidation of ß-hydroxybutyrate both with intact mitochondria (146) and with submitochondrial fractions (75). These observations confirm the conclusion that 3 phosphorylations do occur in the
respiratory chain between DPNH and 0 2 .
F. SITES OF PHOSPHORYLATION
In recent years data has accumulated which permits some localization
of the sites of phosphorylation along the respiratory chain. One site was
definitely located when Friedkin and Lehninger (157) and Judah (164)
observed oxidative phosphorylation during the oxidation of ascorbic acid.
In mitochondrial suspensions containing cytochrome c, ascorbate reduces
the heme-protein nonenzymically. Reoxidation of ferrocytochrome c
aerobically results in phosphorylation. Maley and Lardy (165) and
Lehninger et al. (166) have obtained P/O ratios approaching 1.0 for
this reaction with rat liver mitochondria. Since ascorbate is a nonspecific
reductant for cytochrome c, other reducing agents should substitute
for ascorbate. The authors report that cysteine and adrenaline would
substitute, but that lower P/O ratios were observed. This could be due
to the fact that oxidation products of these compounds inhibit phosphorylation.
465
Other workers have demonstrated that the oxidation of isocitrate by
a TPN
+ -linked dehydrogenase in rat liver mitochondria proceeds with a
P/O ratio of approximately 1.0 (163). The evidence indicates that the
single phosphorylation step occurs when ferrocytochrome c, reduced in
the presence of TPNH and cytochrome c reductase, is oxidized by molecular 0 2 . This mechanism provides a means for obtaining some of the
potential energy available in TPNH, but the process is much less efficient
energetically than the process in which DPN
+ is reduced through the
enzyme transhydrogenase.
3. Oxidation of ß-Hydroxybutyrate
Before highly purified DPNH was available, Lehninger's group utilized the oxidation of ß-hydroxybutyrate to obtain information concerning the phosphorylation processes coupled to pyridine nucleotide oxidations (159). The oxidation of ß-hydroxybutyrate is a single step oxidation catalyzed by a DPN
+ -requiring dehydrogenase. One of the products
formed, acetoacetate, was metabolically inert in their system. The DPNH
formed was reoxidized by the respiratory chain. Various factors
(Mg
2+ , arsenite, dinitrophenol) which were known to influence oxidative
phosphorylation with DPNH also influenced phosphorylation associated
with /?-hydroxybutyrate to the same extent. There is no evidence to indicate that phosphorylation might occur at the substrate level between
substrate and DPN
+ (145). P/O ratios in excess of 2.0 have been
obtained in the oxidation of ß-hydroxybutyrate both with intact mitochondria (146) and with submitochondrial fractions (75). These observations confirm the conclusion that 3 phosphorylations do occur in the
respiratory chain between DPNH and 0 2 .
F. SITES OF PHOSPHORYLATION
In recent years data has accumulated which permits some localization
of the sites of phosphorylation along the respiratory chain. One site was
definitely located when Friedkin and Lehninger (157) and Judah (164)
observed oxidative phosphorylation during the oxidation of ascorbic acid.
In mitochondrial suspensions containing cytochrome c, ascorbate reduces
the heme-protein nonenzymically. Reoxidation of ferrocytochrome c
aerobically results in phosphorylation. Maley and Lardy (165) and
Lehninger et al. (166) have obtained P/O ratios approaching 1.0 for
this reaction with rat liver mitochondria. Since ascorbate is a nonspecific
reductant for cytochrome c, other reducing agents should substitute
for ascorbate. The authors report that cysteine and adrenaline would
substitute, but that lower P/O ratios were observed. This could be due
to the fact that oxidation products of these compounds inhibit phosphorylation.