Reversible Thiol Oxidation Inhibits the Mitochondrial ATP Synthase in <i>Xenopus Laevis</i> Oocytes

• Main Authors: James Cobley, Anna Noble, Rachel Bessell, Matthew Guille, Holger Husi
• Format: Article
• Language: English
• Published: MDPI AG 2020-03-01
• Series: Antioxidants
• Subjects: mitochondria; thiol; redox signaling; atp synthase; oocyte; xenopus laevis; click chemistry
• Online Access: https://www.mdpi.com/2076-3921/9/3/215

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^&#8722;¹/mg^&#8722;¹) 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 F₁ (e.g., ATP-&#945;-F₁) and F_o (e.g., subunit c) are reversibly oxidised. Catalyst-free TCO-Tz Click PEGylation reveals significant (~60%) reversible ATP-&#945;-F₁oxidation at two evolutionary conserved cysteine residues (C²⁴⁴ and C²⁹⁴) 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^&#8722;¹/mg^&#8722;¹in oocytes. We conclude that reversible thiol oxidation inhibits the mitochondrial ATP synthase in <i>X. laevis</i> oocytes.