Two protons are pumped from the mitochondrial matrix per electron transferred between NADH and ubiquinone

The spectrophotometric indicators neutral red and safranine were used to determine the relative H+/e- ratios of proton uptake from the mitochondrial matrix, and the q+/e- ratios of electrical charge translocation, during oxidation of beta-OH-butyrate and succinate by ferricyanide in rat liver mitochondria. With beta-OH-butyrate both ratios were higher than with succinate by a factor close to 3.0. Since there is full agreement that H+/e- of proton uptake and q+/e- of charge translocation are both equal to unity for oxidation of succinate (or ubiquinol) by ferricytochrome c, the corresponding ratios for oxidation of NADH by ubiquinone and cytochrome c are near 2.0 and 3.0, respectively.