Catalytic mechanism of cofactor-free dioxygenases and how they circumvent spin-forbidden oxygenation of their substrates.

Research output: Contribution to journalArticle

  • External authors:
  • Aitor Hernández-Ortega
  • Matthew Quesne
  • Soi Bui
  • Derren J. Heyes
  • Roberto A. Steiner

Abstract

Dioxygenases catalyze a diverse range of biological reactions by incorporating molecular oxygen into organic substrates. Typically, they use transition metals or organic cofactors for catalysis. Bacterial 1-H-3-hydroxy-4-oxoquinaldine-2,4-dioxygenase (HOD) catalyzes the spin-forbidden transfer of dioxygen to its N-heteroaromatic substrate in the absence of any cofactor. We combined kinetics, spectroscopic and computational approaches to establish a novel reaction mechanism. The present work gives insight into the rate limiting steps in the reaction mechanism, the effect of first-coordination sphere amino acids as well as electron-donating/electron-withdrawing substituents on the substrate. We highlight the role of active site residues Ser101/Trp160/His251 and their involvement in the reaction mechanism. The work shows, for the first time, that the reaction is initiated by triplet dioxygen and its binding to deprotonated substrate and only thereafter a spin state crossing to the singlet spin state occurs. As revealed by steady- and transient-state kinetics the oxygen-dependent steps are rate-limiting, whereas Trp160 and His251 are essential residues for catalysis and contribute to substrate positioning and activation, respectively. Computational modeling further confirms the experimental observations and rationalizes the electron transfer pathways, and the effect of substrate and substrate binding pocket residues. Finally, we make a direct comparison with iron-based dioxygenases and explain the mechanistic and electronic differences with cofactor-free dioxygenases. Our multidisciplinary study confirms that the oxygenation reaction can take place in absence of any cofactor by a unique mechanism in which the specially designed fit-for-purpose active-site architecture modulates substrate reactivity toward oxygen.

Bibliographical metadata

Original languageEnglish
Article number10.1021/jacs.5b03836
Pages (from-to)7474-7487
Number of pages13
JournalJournal of the American Chemical Society
Volume137
DOIs
StatePublished - 19 May 2015