P450 CYP17A1 Variant with a Disordered Proton Shuttle Assembly Retains Peroxo-Mediated Lyase Efficiency

Yilin Liu, Ilia G. Denisov, Yelena V. Grinkova, Stephen G. Sligar, James R. Kincaid

Research output: Contribution to journalArticlepeer-review


Human cytochrome P450 CYP17A1 first catalyzes hydroxylation at the C17 position of either pregnenolone (PREG) or progesterone (PROG), and a subsequent C17−C20 bond scission to produce dehydroepiandrosterone (DHEA) or androstenedione (AD). In the T306A mutant, replacement of the Threonine 306 alcohol functionality, essential for efficient proton delivery in the hydroxylase reaction, has only a small effect on the lyase activity. In this work, resonance Raman spectroscopy is employed to provide crucial structural insight, confirming that this mutant, with its disordered proton shuttle, fails to generate essential hydroxylase pathway intermediates, accounting for the loss in hydroxylase efficiency. Significantly, a corresponding spectroscopic study with the susceptible lyase substrate, 17-OH PREG, not only reveals an initially trapped peroxo-iron intermediate experiencing an H-bond interaction of the 17-OH group with the proximal oxygen of the Fe-Op-Ot fragment, facilitating peroxo- attack on the C20 carbon, but also unequivocally shows the presence of the subsequent hemiketal intermediate of the lyase reaction.

Original languageEnglish (US)
Pages (from-to)16846-16852
Number of pages7
JournalChemistry - A European Journal
Issue number70
StatePublished - Dec 15 2020


  • P450 CYP17A1
  • T306A mutant
  • cryoradiolysis
  • proton shuttle
  • resonance Raman spectroscopy

ASJC Scopus subject areas

  • Catalysis
  • Organic Chemistry


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