466
ERIC E. CONN
More recently, Nielsen and Lehninger (153) have re-examined the
amount of phosphorylation coupled to the oxidation of ferrocytochrome
c. Ferrocytochrome c was used in stoichiometric amounts rather than
in catalytic quantities. As a result, methods were employed for measuring millimicromolar amounts of esterified phosphate and cytochrome.
In these experiments the highest P/O ratio was 0.86, in good agreement with earlier values. In experiments of this type, the P/O ratio is
equal to the expression:
mMmoles phosphate taken up
0.5 mjumoles ferrocytochrome c oxidized
Similar values were obtained using a submitochondrial fraction from
rat liver particles (76). For some time Slater questioned the existence
of a phosphorylation step between cytochrome c and oxygen (155), but
this has now been conceded (167).
With regard to the region of the respiratory chain between DPNH
and cytochrome c it was known that phosphorylation occurred when
ferricytochrome c (168) or ferricyanide was the oxidizing agent (141,
146, 164). The point at which ferricyanide reacts with the chain is not
known, but Copenhaver and Lardy concluded that at least one phosphorylation occurred between the substrate and the point in the respiratory chain which is sensitive to antimycin A. Again using methods for
measuring millimicromolar amounts of esterified phosphate, Borgström
(168a) measured the phosphorylation which occurred during the oxidation of ß-hydroxybutyrate by ferricytochrome c. The reduction of ferricytochrome c was measured and its reoxidation prevented by the presence of cyanide in the reaction mixture. The average P/O ratio observed
was 1.51, confirming the maximum value of 2.0 expected. In later studies with submitochondrial fractions, Devlin and Lehninger (49) observed P/O ratios no greater than 1.0 for the oxidation of /?-hydroxybutyrate by ferricytochrome c and concluded that this lower value represented a partial failure of the phosphorylation mechanism in these
small particles.
In summarizing the preceding material, it appears that two phosphorylation steps occur somewhere between DPNH and cytochrome c
and that one exists between cytochrome c and 0 2 . However, if the cytochrome respiratory path has the composition shown in Scheme V, more
precise localization is desirable. Chance and Williams (SI, 169) have
discussed their approach to this problem and have defined "cross over
points" as points in the respiratory chain where, as a result of some
experimental condition, all of the carriers on one side of the point become oxidized and all of the carriers on the other are reduced. For
example, when antimycin A is added to respiring mitochondria, cyto-
ERIC E. CONN
More recently, Nielsen and Lehninger (153) have re-examined the
amount of phosphorylation coupled to the oxidation of ferrocytochrome
c. Ferrocytochrome c was used in stoichiometric amounts rather than
in catalytic quantities. As a result, methods were employed for measuring millimicromolar amounts of esterified phosphate and cytochrome.
In these experiments the highest P/O ratio was 0.86, in good agreement with earlier values. In experiments of this type, the P/O ratio is
equal to the expression:
mMmoles phosphate taken up
0.5 mjumoles ferrocytochrome c oxidized
Similar values were obtained using a submitochondrial fraction from
rat liver particles (76). For some time Slater questioned the existence
of a phosphorylation step between cytochrome c and oxygen (155), but
this has now been conceded (167).
With regard to the region of the respiratory chain between DPNH
and cytochrome c it was known that phosphorylation occurred when
ferricytochrome c (168) or ferricyanide was the oxidizing agent (141,
146, 164). The point at which ferricyanide reacts with the chain is not
known, but Copenhaver and Lardy concluded that at least one phosphorylation occurred between the substrate and the point in the respiratory chain which is sensitive to antimycin A. Again using methods for
measuring millimicromolar amounts of esterified phosphate, Borgström
(168a) measured the phosphorylation which occurred during the oxidation of ß-hydroxybutyrate by ferricytochrome c. The reduction of ferricytochrome c was measured and its reoxidation prevented by the presence of cyanide in the reaction mixture. The average P/O ratio observed
was 1.51, confirming the maximum value of 2.0 expected. In later studies with submitochondrial fractions, Devlin and Lehninger (49) observed P/O ratios no greater than 1.0 for the oxidation of /?-hydroxybutyrate by ferricytochrome c and concluded that this lower value represented a partial failure of the phosphorylation mechanism in these
small particles.
In summarizing the preceding material, it appears that two phosphorylation steps occur somewhere between DPNH and cytochrome c
and that one exists between cytochrome c and 0 2 . However, if the cytochrome respiratory path has the composition shown in Scheme V, more
precise localization is desirable. Chance and Williams (SI, 169) have
discussed their approach to this problem and have defined "cross over
points" as points in the respiratory chain where, as a result of some
experimental condition, all of the carriers on one side of the point become oxidized and all of the carriers on the other are reduced. For
example, when antimycin A is added to respiring mitochondria, cyto-