Abstract
Superoxide is continuously removed from cells by superoxide dismutase (SOD). Genetic knockout studies have shown that SOD is crucial for survival, suggesting that higher levels of superoxide are damaging. Tyrosyl radicals are a likely target because they react rapidly with superoxide, either by reduction to form tyrosine (repair), or by oxidative addition to form reactive hydroperoxides. Because tyrosyl radicals are formed on proteins under pathological and physiological conditions, taking a closer at look these reactions will shed light on the role of superoxide and SOD in aerobic organisms. The purpose of this thesis was to investigate the reaction between superoxide and protein tyrosyl radicals generated by different mechanisms.
The reaction of hydrogen peroxide with sperm whale myoglobin is a useful model because tyrosyl radicals are produced, facilitating myoglobin dimer formation. When myoglobin was treated with hydrogen peroxide and superoxide, dimer formation was greatly decreased. SOD restored dimer formation in a dose dependent manner. Tryptic digestion of products, analysed by mass spectrometry, revealed evidence for repair and addition, with addition occurring specifically on Tyr151. Overall, the ratio of repair to addition was approximately 10:1. These results show that superoxide is capable of reacting with tyrosyl radicals formed on proteins, with tyrosine hydroperoxide and tyrosine hydroxide as potential products.
To detect superoxide addition products in biological samples, other members of my host lab began antibody development. Attempts to produce sufficient quantities of tyrosine hydroxide as an antigen led to the discovery that glutathione conjugates readily to this product. My initial attempts to detect the glutathione adduct on oxidised myoglobin via mass spectrometry and immunoblotting revealed that the adduct was reversible when excess glutathione was removed. After treatment with sodium borohydride the glutathione adduct was detected in tryptic digest samples as well as via anti-GSH immunoblotting. These results suggest that any tyrosine hydroxide arising in vivo should be conjugated to thiols, with implications for protein aggregation and cell signalling.
During oxidative stress, the transfer of radicals from free tyrosine to proteins can occur. Using insulin aspart as a model protein, free tyrosine transferred radical equivalents to the protein when oxidation was initiated by a peroxidase. In the absence of superoxide, a number of dityrosine products formed as measured by mass spectrometry. Superoxide prevented the formation of these dityrosine products, and addition products were detected, both on the whole protein as well as in tryptic digests. Superoxide addition was localised to Tyr14. These results suggest that tyrosine hydroperoxide formation on proteins may occur under conditions of oxidative stress that involve peroxidases and free tyrosine.
Ribonucleotide reductase (RNR) catalyses the rate-limiting step of DNA synthesis, with catalysis requiring radical transfer along a pathway of tyrosines. Superoxide has been shown to remove the radical signal in vitro, and increase the mass of the whole protein corresponding to superoxide addition. Therefore, RNR activity was measured in sodΔ strains of S. cerevisiae. RNR activity in cell lysate from both sod2Δ and sod1Δ cells was decreased compared to wt. When superoxide production was increased with the paraquat treatment, sod1Δ cells were the most sensitive with respect to RNR activity loss and decreased growth. These results suggest that SOD is important for maintaining RNR activity.
In summary, the findings in this thesis support the hypothesis that the reactions of superoxide with protein tyrosyl radicals could contribute to superoxide toxicity. They also show that SOD has the potential to mitigate this damage.