Effects of Inhibiting CoQ10 Biosynthesis with 4-nitrobenzoate in Human Fibroblasts

Coenzyme Q10 (CoQ10) is a potent lipophilic antioxidant in cell membranes and a carrier of electrons in the mitochondrial respiratory chain. We previously characterized the effects of varying severities of CoQ10 deficiency on ROS production and mitochondrial bioenergetics in cells harboring genetic defects of CoQ10 biosynthesis. We observed a unimodal distribution of ROS production with CoQ10 deficiency: cells with <20% of CoQ10 and 50–70% of CoQ10 did not generate excess ROS while cells with 30–45% of CoQ10 showed increased ROS production and lipid peroxidation. Because our previous studies were limited to a small number of mutant cell lines with heterogeneous molecular defects, here, we treated 5 control and 2 mildly CoQ10 deficient fibroblasts with varying doses of 4-nitrobenzoate (4-NB), an analog of 4-hydroxybenzoate (4-HB) and inhibitor of 4-para-hydroxybenzoate:polyprenyl transferase (COQ2) to induce a range of CoQ10 deficiencies. Our results support the concept that the degree of CoQ10 deficiency in cells dictates the extent of ATP synthesis defects and ROS production and that 40–50% residual CoQ10 produces maximal oxidative stress and cell death

Yeast Coq9 controls deamination of coenzyme Q intermediates that derive from para-aminobenzoic acid

Coq9 is a polypeptide subunit in a mitochondrial multi-subunit complex, termed the CoQ-synthome, required for biosynthesis of coenzyme Q (ubiquinone or Q). Deletion of COQ9 results in dissociation of the CoQ-synthome, but over-expression of Coq8 putative kinase stabilizes the CoQ-synthome in the coq9 null mutant and leads to the accumulation of two nitrogen containing Q-intermediates, imino-demethoxy-Q(6) (IDMQ(6)) and 3-hexaprenyl-4-aminophenol (4-AP) when para-aminobenzoic acid (pABA) is provided as a ring precursor. To investigate whether Coq9 is responsible for deamination steps in Q biosynthesis, we utilized the yeast coq5-5 point mutant. The yeast coq5-5 point mutant is defective in the C-methyltransferase step of Q biosynthesis, but retains normal steady-state levels of the Coq5 polypeptide. Here we show that when high amounts of (13)C(6)-pABA are provided, the coq5-5 mutant accumulates both (13)C(6)-imino-demethyl-demethoxy-Q(6) ((13)C(6)-IDDMQ(6)) and demethyl-demethoxy-Q(6) ((13)C(6)-DDMQ(6)). Deletion of COQ9 in the yeast coq5-5 mutant along with Coq8 over-expression and (13)C(6)-pABA labeling leads to the absence of (13)C(6)-DDMQ(6), and the nitrogen-containing intermediates (13)C(6)-4-AP and (13)C(6)-IDDMQ(6) persist. We describe a coq9 temperature sensitive mutant and show that at the non-permissive temperature, steady state polypeptide levels of Coq9-ts19 increased, while Coq4, Coq5, Coq6, and Coq7 decreased. The coq9-ts19 mutant had decreased Q(6) content and increased levels of nitrogen-containing intermediates. These findings identify Coq9 as a multi-functional protein that is required for the function of Coq6 and Coq7 hydroxylases, for removal of the nitrogen substituent from pABA-derived Q-intermediates, and is an essential component of the CoQ synthome