Stoichiometry of H + ejection coupled to electron transport through site 2 in ascites tumor mitochondria

The stoichiometry of H + ejection coupled to electron flow from succinate to ferricyanide in the electron transport chain of mitochondria from Ehrlich ascites tumor and AS30-D hepatoma cells was determined. Values close to 4.0 for the H + 2e − ejection ratio were found in both cell lines, with either Ca 2+ or K + (+ valinomycin) as charge-compensating permeant cation. The 4 H + ejected were compensated by outward movement of two negative charges to reduce 2 Fe(CN)6 3− to 2 Fe(CN)6 4−, and the uptake of two positive charges in the form of the permeant cation. Experiments on (a) omission of rotenone (b) the effect of antimycin A and (c) depletion of endogenous NAD(P)-linked substrates showed that no significant endogenous electron flow or H + ejection occurred, thus eliminating possible overestimation of the H +/2e − ratio from endogenous substrates. These data on mitochondria from two tumor cell lines are fully consistent with earlier measurements of the H +/O stoichiometry for succinate and NADH oxidation in tumor mitochondria and with the H + 2e − stoichiometry for site 2 in normal rat liver mitochondria.