Conversion of 3Fe-4S to 4Fe-4S clusters in native pyruvate formate-lyase activating enzyme: Mössbauer characterization and implications for mechanism

C. Krebs, T. F. Henshaw, J. Cheek, B. H. Huynh, J. B. Broderick

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Abstract

Pyruvate formate-lyase activating enzyme utilizes an iron-sulfur cluster and S-adenosylmethionine to generate the catalytically essential glycyl radical on pyruvate formate-lyase. Variable-temperature (4.2-200 K) and variable-field (0.05-8 T) Mössbauer spectroscopy has been used to characterize the iron-sulfur clusters present in anaerobically isolated pyruvate formate-lyase activating enzyme and in the dithionite-reduced form of the enzyme. Detailed analysis of the Mössbauer data indicates that the anaerobically isolated enzyme contains a mixture of Fe-S clusters with the cuboidal [3Fe-4S]+ clusters as the primary cluster form, accounting for 66% of the total iron. Other forms present include [2Fe-2S]2+ (12% of total Fe) and [4Fe-4S]2+ (8% of total iron). Careful examination of Mössbauer spectra recorded at various applied fields reveal a fourth spectral component which is assigned to a linear [3Fe-4S]+ (∼10% of total Fe). Reduction of the as-isolated enzyme by dithionite, interestingly, converts all cluster types into the [4Fe-4S] form with a mixture of 2+ (66% of total iron) and 1 + (12% of total iron) oxidation states. These results are discussed in light of the proposed role for the iron-sulfur cluster in radical generation.

Original languageEnglish (US)
Pages (from-to)12497-12506
Number of pages10
JournalJournal of the American Chemical Society
Volume122
Issue number50
DOIs
StatePublished - Dec 20 2000

All Science Journal Classification (ASJC) codes

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

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