| Summary: | Oocytes are postulated to repress the proton pumps (e.g., complex IV) and ATP synthase to safeguard mitochondrial DNA homoplasmy by curtailing superoxide production. Whether the ATP synthase is inhibited is, however, unknown. Here we show that: oligomycin sensitive ATP synthase activity is significantly greater (~170 vs. 20 nmol/min<sup>−</sup><sup>1</sup>/mg<sup>−</sup><sup>1</sup>) in testes compared to oocytes in <i>Xenopus laevis </i>(<i>X. laevis</i>). Since ATP synthase activity is redox regulated, we explored a regulatory role for reversible thiol oxidation. If a protein thiol inhibits the ATP synthase, then constituent subunits must be reversibly oxidised. Catalyst-free <i>trans</i>-cyclooctene 6-methyltetrazine (TCO-Tz) immunocapture coupled to redox affinity blotting reveals several subunits in F<sub>1</sub> (e.g., ATP-α-F<sub>1</sub>) and F<sub>o</sub> (e.g., subunit c) are reversibly oxidised. Catalyst-free TCO-Tz Click PEGylation reveals significant (~60%) reversible ATP-α-F<sub>1 </sub>oxidation at two evolutionary conserved cysteine residues (C<sup>244</sup> and C<sup>294</sup>) in oocytes. TCO-Tz Click PEGylation reveals ~20% of the total thiols in the ATP synthase are substantially oxidised. Chemically reversing thiol oxidation significantly increased oligomycin sensitive ATP synthase activity from ~12 to 100 nmol/min<sup>−</sup><sup>1</sup>/mg<sup>−</sup><sup>1 </sup>in oocytes. We conclude that reversible thiol oxidation inhibits the mitochondrial ATP synthase in <i>X. laevis</i> oocytes.
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