The total biosynthesis of ubiquinone-9 from p-hydroxy[U-¹⁴C]benzoate has been demonstrated in mitochondria isolated from rat liver slices preincubated with mevalonic acid. Two partial reactions were also demonstrated. These were the conversion of p-hydroxybenzoate to 5-demethoxyubiquinone-9 and sequentially, the conversion of 5-demethoxyubiquinone-9 to ubiquinone-9. Furthermore, these reactions were found to be catalyzed by the inner membrane of the mitochondrion.

It appeared that the only external requirements for this mitochondrial system are ATP, p-hydroxybenzoate, and a source of isopentenyl pyrophosphate. Conversion of 5-demethoxyubiquinone-9 to ubiquinone-9, which involves one hydroxylation and one methylation, is dependent upon molecular oxygen, S-adenosylmethionine, and reducing equivalents as indicated by a preference for succinate over malate. The over-all reaction is inhibited by CO but not by KCN. External ATP was essential for the conversion of p-hydroxybenzoate to 5-demethoxyubiquinone-9, but not for the conversion of 5-demethoxyubiquinone-9 to ubiquinone-9. These data suggest that the inner membrane of liver mitochondria is capable of the hydroxylation and methylation of aromatic precursors of ubiquinone.