Catalyst-free Click PEGylation reveals substantial mitochondrial ATP synthase sub-unit alpha oxidation before and after fertilisation

James Cobley, Holger Husi, Eduardo Jimenez Fernandez

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15 Citations (Scopus)
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Using non-reducing Western blotting to assess protein thiol redox state is challenging because most reduced and oxidised forms migrate at the same molecular weight and are, therefore, indistinguishable. While copper catalysed Click chemistry can be used to ligate a polyethylene glycol (PEG) moiety termed Click PEGylation to mass shift the reduced or oxidised form as desired, the potential for copper catalysed auto-oxidation is problematic. Here we define a catalyst-free transcyclooctene-methyltetrazine (TCO-Tz) inverse electron demand Diels Alder chemistry approach that affords rapid (k ~2000 M-1 s-1), selective and bio-orthogonal Click PEGylation. We used TCO-Tz Click PEGylation to investigate how fertilisation impacts reversible mitochondrial ATP synthase F1-Fo sub-unit alpha (ATP-α-F1) oxidation—an established molecular correlate of impaired enzyme activity—in Xenopus laevis. TCO-Tz Click PEGylation studies reveal substantial (~65%) reversible ATP-α-F1 oxidation at evolutionary conserved cysteine residues (i.e., C244 and C294) before and after fertilisation. A single thiol is, however, preferentially oxidised likely due to greater solvent exposure during the catalytic cycle. Selective reduction experiments show that: S-glutathionylation accounts for ~50-60% of the reversible oxidation observed, making it the dominant oxidative modification type. Intramolecular disulphide bonds may also contribute due to their relative stability. Substantial reversible ATP-α-F1 oxidation before and after fertilisation is biologically meaningful because it implies low mitochondrial F1-Fo ATP synthase activity. Catalyst-free TCO-Tz Click PEGylation is a valuable new tool to interrogate protein thiol redox state in health and disease.
Original languageEnglish
Article number101258
Pages (from-to)1-8
Number of pages8
JournalRedox Biology
Early online date18 Jun 2019
Publication statusE-pub ahead of print - 18 Jun 2019


  • Mitochondria
  • ATP synthase
  • Fertilisation
  • Development
  • Redox signalling


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