No abstract

The facile abstraction of bis-allylic hydrogens from polyunsaturated fatty acids (PUFAs) is the hallmark chemistry responsible for initiation and propagation of autoxidation reactions. The products of these autoxidation reactions can form cross-links to other membrane components, damage proteins and nucleic acid. We report that PUFAs deuterated at bis-allylic sites are much more resistant to autoxidation reactions, due to the isotope effect. This is shown using coenzyme Q-deficient Saccharomyces cerevisiae coq mutants with defects in biosynthesis of coenzyme Q (Q). Q functions in respiratory energy metabolism and also functions as a lipid-soluble antioxidant. Yeast coq mutants incubated in the presence of the PUFAs α-linolenic or linoleic acid exhibit 99% loss of colony formation after four hours, demonstrating a profound loss of viability. In contrast, coq mutants treated with monounsaturated oleic acid or with one of the deuterated PUFAs:11,11-D2-Linoleic or 11,11,14,14-D4-αLinolenic retain viability similar to wild-type yeast. Deuterated PUFAs also confer protection to wild-type yeast subjected to heat stress. These results indicate that isotope-reinforced PUFAs are stabilized compared to standard PUFAs, and they protect coq mutants and wild-type yeast cells against the toxic effects of lipid autoxidation products. These findings suggest new approaches to controlling ROS-inflicted cellular damage and oxidative stress.

Coenzyme Qn (ubiquinone or Qn) is a redox active lipid composed of a fully substituted benzoquinone ring and a polyisoprenoid tail of n isoprene units. Saccharomyces cerevisiae coq1-coq9 mutants have defects in Q biosynthesis, lack Q6, are respiratory defective, and sensitive to stress imposed by polyunsaturated fatty acids. The hallmark phenotype of the Q-less yeast coq mutants is that respiration in isolated mitochondria can be rescued by the addition of Q2, a soluble Q analog. Yeast coq10 mutants share each of these phenotypes, with the surprising exception that they continue to produce Q6. Structure determination of the Caulobacter crescentus Coq10 homolog (CC1736) revealed a steroidogenic acute regulatory protein-related lipid transfer (START) domain, a hydrophobic tunnel known to bind specific lipids in other START domain family members. Here we show that purified CC1736 binds Q2, Q3, Q10, or demethoxy-Q3 in an equimolar ratio, but fails to bind 3-farnesyl-4-hydroxybenzoic acid, a farnesylated analog of an early Q-intermediate. Over-expression of C. crescentus CC1736 or COQ8 restores respiratory electron transport and antioxidant function of Q6 in the yeast coq10 null mutant. Studies with stable isotope ring precursors of Q reveal that early Q-biosynthetic intermediates accumulate in the coq10 mutant and de novo Q-biosynthesis is less efficient than in the wild-type yeast or rescued coq10 mutant. The results suggest that the Coq10 polypeptide:Q (protein:ligand) complex may serve essential functions in facilitating de novo Q biosynthesis and in delivering newly synthesized Q to one or more complexes of the respiratory electron transport chain.

The facile abstraction of bis-allylic hydrogens from polyunsaturated fatty acids (PUFAs) is the hallmark chemistry responsible for initiation and propagation of autoxidation reactions. The products of these autoxidation reactions can form cross-links to other membrane components, damage proteins and nucleic acid. We report that PUFAs deuterated at bis-allylic sites are much more resistant to autoxidation reactions, due to the isotope effect. This is shown using coenzyme Q-deficient Saccharomyces cerevisiae coq mutants with defects in biosynthesis of coenzyme Q (Q). Q functions in respiratory energy metabolism and also functions as a lipid-soluble antioxidant. Yeast coq mutants incubated in the presence of the PUFAs α-linolenic or linoleic acid exhibit 99% loss of colony formation after four hours, demonstrating a profound loss of viability. In contrast, coq mutants treated with monounsaturated oleic acid or with one of the deuterated PUFAs:11,11-D 2 -Linoleic or 11,11,14,14-D 4 -αLinolenic retain viability similar to wild-type yeast. Deuterated PUFAs also confer protection to wild-type yeast subjected to heat stress. These results indicate that isotope-reinforced PUFAs are stabilized compared to standard PUFAs, and they protect coq mutants and wild-type yeast cells against the toxic effects of lipid autoxidation products. These findings suggest new approaches to controlling ROS-inflicted cellular damage and oxidative stress.

No abstract

Polyunsaturated fatty acids (PUFAs) are exquisitely sensitive to autoxidation damage. The enhanced vulnerability of PUFAs to such autoxidation stems from the labile nature of the bis‐allylic hydrogen atoms. PUFAs synthesized to contain Deuterium atoms uniquely at the bis‐allylic sites (termed isotope‐reinforced PUFAs) would be expected to be more resistant to autoxidation reactions due to the isotope effect. This hypothesis was tested by making use of yeast coq mutants with defects in biosynthesis of coenzyme Q (CoQ, or ubiquinone). CoQ plays a well‐known role in respiratory energy metabolism and also functions as a lipid soluble chain terminating antioxidant. Although yeast cannot synthesize PUFAs, they are able to incorporate exogenously supplied PUFAs. Yeast coq mutants incubated in the presence of linolenic acid (C18:3) exhibit profound loss of viability as ascertained by greater than 99% loss of colony formation at 4 hours. In contrast, the coq mutants treated with the isotope‐reinforced linolenic acid (bis‐allylic D4‐C18:3) retain 80–90% viability, a value similar to wild‐type or CoQ‐replete yeast. These results indicate that isotope reinforced PUFAs are stabilized as compared to standard PUFAs, and the coq mutant yeast cells containing the D4‐linolenic acid are protected against the toxic effects of lipid autoxidation products. This research was supported by NIH GM45952.