Salicylate 5-Hydroxylase: Intermediates in Aromatic Hydroxylation by a Rieske Monooxygenase

Melanie S. Rogers, John D. Lipscomb

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

Rieske oxygenases (ROs) catalyze a large range of oxidative chemistry. We have shown that cis-dihydrodiol-forming Rieske dioxygenases first react with their aromatic substrates via an active site nonheme Fe(III)-superoxide; electron transfer from the Rieske cluster then completes the product-forming reaction. Alternatively, two-electron-reduced Fe(III)-peroxo or hydroxo-Fe(V)-oxo activated oxygen intermediates are possible and may be utilized by other ROs to expand the catalytic range. Here, the reaction of a Rieske monooxygenase, salicylate 5-hydroxylase, that does not form a cis-dihydrodiol is examined. Single-turnover kinetic studies show fast binding of salicylate and O2. Transfer of the Rieske electron required to form the gentisate product occurs through bonds over â12 Å and must also be very fast. However, the observed rate constant for this reaction is much slower than expected and sensitive to substrate type. This suggests that initial reaction with salicylate occurs using the same Fe(III)-superoxo-level intermediate as Rieske dioxygenases and that this reaction limits the observed rate of electron transfer. A transient intermediate (λmax = 700 nm) with an electron paramagnetic resonance (EPR) at g = 4.3 is observed after the product is formed in the active site. The use of 17O2 (I = 5/2) results in hyperfine broadening of the g = 4.3 signal, showing that gentisate binds to the mononuclear iron via its C5-OH in the intermediate. The chromophore and EPR signal allow study of product release in the catalytic cycle. Comparison of the kinetics of single- A nd multiple-turnover reactions shows that re-reduction of the metal centers accelerates product release â300-fold, providing insight into the regulatory mechanism of ROs.

Original languageEnglish (US)
Pages (from-to)5305-5319
Number of pages15
JournalBiochemistry
Volume58
Issue number52
DOIs
StatePublished - Dec 31 2019

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural

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