Are free radicals involved in IspH catalysis? An EPR and crystallographic investigation

Weixue Wang, Ke Wang, Ingrid Span, Johann Jauch, Adelbert Bacher, Michael Groll, Eric Oldfield

Research output: Contribution to journalArticle

Abstract

The [4Fe-4S] protein IspH in the methylerythritol phosphate isoprenoid biosynthesis pathway is an important anti-infective drug target, but its mechanism of action is still the subject of debate. Here, by using electron paramagnetic resonance (EPR) spectroscopy and 2H, 17O, and 57Fe isotopic labeling, we have characterized and assigned two key reaction intermediates in IspH catalysis. The results are consistent with the bioorganometallic mechanism proposed earlier, and the mechanism is proposed to have similarities to that of ferredoxin, thioredoxin reductase, in that one electron is transferred to the [4Fe-4S] 2+ cluster, which then performs a formal two-electron reduction of its substrate, generating an oxidized high potential iron-sulfur protein (HiPIP)-like intermediate. The two paramagnetic reaction intermediates observed correspond to the two intermediates proposed in the bioorganometallic mechanism: the early π-complex in which the substrate's 3-CH 2OH group has rotated away from the reduced iron-sulfur cluster, and the next, η 3-allyl complex formed after dehydroxylation. No free radical intermediates are observed, and the two paramagnetic intermediates observed do not fit in a Birch reduction-like or ferraoxetane mechanism. Additionally, we show by using EPR spectroscopy and X-ray crystallography that two substrate analogues (4 and 5) follow the same reaction mechanism.

Original languageEnglish (US)
Pages (from-to)11225-11234
Number of pages10
JournalJournal of the American Chemical Society
Volume134
Issue number27
DOIs
StatePublished - Jul 11 2012

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry

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