Spectroscopic and electronic structure studies of intermediate X in ribonucleotide reductase R2 and two variants: A description of the Fe IV-oxo bond in the FeIII-O-FeIV dimer

Nataša Mitić, Michael D. Clay, Lana Saleh, Joseph M. Bollinger, Jr., Edward I. Solomon

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Spectroscopic and electronic structure studies of the class I Escherichia coli ribonucleotide reductase (RNR) intermediate X and three computationally derived model complexes are presented, compared, and evaluated to determine the electronic and geometric structure of the FeIII-FeIV active site of intermediate X. Rapid freeze-quench (RFQ) EPR, absorption, and MCD were used to trap intermediate X in R2 wild-type (WT) and two variants, W48A and Y122F/Y356F. RFQ-EPR spin quantitation was used to determine the relative contributions of intermediate X and radicals present, while RFQ-MCD was used to specifically probe the FeIII/FeIV active site, which displayed three FeIV d-d transitions between 16 700 and 22 600 cm-1, two FeIV d-d spin-flip transitions between 23 500 and 24 300 cm-1, and five oxo to FeIV and FeIII charge transfer (CT) transitions between 25 000 and 32 000 cm-1. The FeIV d-d transitions were perturbed in the two variants, confirming that all three d-d transitions derive from the d-π manifold. Furthermore, the FeIV d-π splittings in the WT are too large to correlate with a bis-μ-oxo structure. The assignment of the FeIV d-d transitions in WT intermediate X best correlates with a bridged μ-oxo/μ-hydroxo [Fe III(μ-O)(μ-OH)FeIV] structure. The μ-oxo/μ-hydroxo core structure provides an important σ/π superexchange pathway, which is not present in the bis-μ-oxo structure, to promote facile electron transfer from Y122 to the remote FeIV through the bent oxo bridge, thereby generating the tyrosyl radical for catalysis.

Original languageEnglish (US)
Pages (from-to)9049-9065
Number of pages17
JournalJournal of the American Chemical Society
Issue number29
Publication statusPublished - Jul 25 2007


All Science Journal Classification (ASJC) codes

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

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