JBC, Vol. 251, Issue 18, 5670-5679, Sep, 1976
Re-evaluation of the H+/site ratio of mitochondrial electron transport with the oxygen pulse technique
M. D. Brand, B. Reynafarje and A. L. Lehninger
The number of protons ejected per pair of electrons passing each
energy-conserving site in the electron transport chain (the H+/site ratio)
has been investigated in rat liver mitochondria by means of the oxygen
pulse technique introduced by Mitchell and Moyle (1967) (Biochem. J. 105,
1147-1162). The usual H+/site values of 2.0 observed by this method were
found to be substantially underestimated as a result of the influx of
phosphate into the mitochondria. This was shown by three different kinds of
experiments. 1. Addition of N-ethylmaleimide or mersalyl, inhibitors of
mitochondrial phosphate transport, increased the H+/site ratio from 2.0 to
3.0. The dependence of this effect on the concentration of either inhibitor
was identical with that for inhibition of phosphate transport. Added
phosphate diminished the H+/site ratio to values below 2.0 in the absence
of N-ethylmaleimide. N-Ethylmaleimide protected the elevated H+/site ratio
of 3.0 against the deleterious effect of added phosphate, but did not
prevent a lowering effect of weak acid anions such as 3-hydroxybutyrate. 2.
Prior washing of mitochondria to remove the endogenous phosphate that leaks
out during the anaerobic preincubation led to H+/site ratios near 3.0,
which were not increased by N-ethylmaleimide. Addition of low
concentrations of phosphate to such phosphate-depleted mitochondria
decreased the H+/site ratio to 2.0; addition of N-ethylmaleimide returned
the ratio to 3.0. 3. Lowering the temperature to 5 degrees, which slows
down phosphate transport, led to H+/site values of 3.0 even in the absence
of N-ethylmaleimide. The H+/site ratio of 3.0 observed in the absence of
phosphate movements was not dependent on any narrowly limited set of
experimental conditions. It occurred with either Ca2+ or K+ (in the
presence of valinomycin) as mobile permeant cation. It was independent of
the concentration of succinate, oxygen, mitochondria, or rotenone,
additions of Ca2+, Li+, or Na+ and was independent of medium pH between 6.5
and 7.5. Inhibitors of the transport of ions or acids other than phosphate
did not affect the H+/site ratio. These results indicate that re-uptake of
endogenous phosphate, lost from mitochondria during anaerobic
preincubation, reduces the observed H+ ejection and leads to underestimated
H+/site ratios of 2.0 in the oxygen pulse method. When phosphate movements
are eliminated by the procedures described above, the observed H+/site
ratio is about 3.0. This value appears to be closer to the true H+/site
ratio for the primary H+ ejection process during electron transport.
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