Identification of protonated oxygenic ligands of ribonucleotide reductase intermediate X

Muralidharan Shanmugam*, Peter E. Doan, Nicholas S. Lees, Jo Anne Stubbe, Brian M. Hoffman

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

7 Scopus citations

Abstract

We previously used a combination of continuous-wave (CW) and pulsed electron-nuclear double resonance (ENDOR) protocols to Identify the types of protonated oxygen (OHx) species and their disposition within the FeIII/FeIV cluster of intermediate X, the direct precursor of the essential diferric-tyrosyl radical cofactor of the β2 subunit of Escherichia coli ribonucleotide reductase (RNR). We concluded that X contains the [(HxO)FeIII OFeIV] fragment (T model), and does not contain α-hydroxo bridge. When combined with a subsequent 17O ENDOR study of X prepared with H2 17O and with 17O2, the results led us to suggest that this fragment Is the entire Inorganic core of X. This has been questioned by recent reports, but these reports do not themselves agree on the core of X. One concluded that X possesses a di-μ-oxo FeIII/FeIV core plus a terminal (H2O) bound to FeIII [e.g., Han, W.-G.; Liu, T.; Lovell, T.; Noodleman, L. J. Am. Chem. Soc. 2005, 127, 15778-15790]. The other [Mitic, N.; Clay, M. D.; Saleh, L.; Bollinger, J. M.; Solomon, E. I. J. Am. Chem. Soc. 2007, 129, 9049-9065] concluded that X contains only a single oxo bridge and postulated the presence of an additional hydroxo bridge plus a terminal hydroxyl bound to FeIII. In this report we take advantage of improvements in 35 GHz pulsed ENDOR performance to reexamine the protonation state of oxygenic ligands of the inorganic core of X by directly probing the exchangeable proton(s) with 2H pulsed ENDOR spectroscopy. These 2H ENDOR measurements confirm that X contains an Fe III-bound terminal aqua ligand (HxO), but the spectra contain none of the features that would be required for the proton of a bridging hydroxyl. Thus, we confirm that X contains a terminal aqua (most likely hydroxo) ligand to FeIII in addition to one or two μ-oxo bridges but does not contain a μ-hydroxo bridge. The 2H ENDOR measurements further demonstrate that this conclusion is applicable to both wild type and Y122F-β2 mutant, and In fact we detect no difference between the properties of protons on the terminal oxygens In the two variants; likewise, 14N ENDOR measurements of hlstidyl ligands bound to Fe show no difference between the two variants.

Original languageEnglish (US)
Pages (from-to)3363-3369
Number of pages7
JournalJournal of the American Chemical Society
Volume131
Issue number9
DOIs
StatePublished - Mar 11 2009

ASJC Scopus subject areas

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

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