The mechanistic stoichiometry for vectorial H+ ejection coupled to electron transport through energy-conserving segments 1 + 2 was determined on
cyanide-inhibited mitochondria from rat liver, rat heart, and
Ehrlich ascites tumor cells, and on rat liver mitoplasts with
ferricyanide or
ferricytochrome c as electron acceptors. K+ (+
valinomycin) and Ca2+ were employed as permeant
cations. Three different methods were employed. In the first, known pulses of
ferricyanide were added, and the total H+ ejected was determined with a glass
electrode. Such measurements gave H+/2e-values exceeding 7.0 for both normal and
tumor mitochondria with
beta-hydroxybutyrate and other
NAD-linked substrates; uptake of Ca2+ was also measured and gave the expected q+/2e-ratios. The second type of measurement was initiated by addition of
ferricytochrome c to rat liver mitoplasts, with H+ ejection monitored with the glass
electrode and
ferricytochrome c reduction by dual-wavelength spectrophotometry; the H+/2e-ratios generally exceeded 7.0. In the third type of measurement, mixing and dilution artifacts were eliminated by oxidizing
ferrocytochrome c in situ with a small amount of
ferricyanide. H+/2e-ratios for rat liver mitoplasts oxidizing
beta-hydroxybutyrate consistently approached or exceeded 7.5. Over 150 measurements made under a variety of conditions gave observed H+/2e-ejection ratios significantly exceeding 7.0, which correlated closely with H+/2e-measurements on sites 1 + 2 + 3, sites 2 + 3, and site 2. Factors leading to the deficit of the observed ratios from the integral value 8 for sites 1 + 2 were discussed.