Function of the diiron cluster of Escherichia coli class Ia ribonucleotide reductase in proton-coupled electron transfer.

Published

Journal Article

The class Ia ribonucleotide reductase (RNR) from Escherichia coli employs a free-radical mechanism, which involves bidirectional translocation of a radical equivalent or "hole" over a distance of ~35 Å from the stable diferric/tyrosyl-radical (Y122(•)) cofactor in the β subunit to cysteine 439 (C439) in the active site of the α subunit. This long-range, intersubunit electron transfer occurs by a multistep "hopping" mechanism via formation of transient amino acid radicals along a specific pathway and is thought to be conformationally gated and coupled to local proton transfers. Whereas constituent amino acids of the hopping pathway have been identified, details of the proton-transfer steps and conformational gating within the β sununit have remained obscure; specific proton couples have been proposed, but no direct evidence has been provided. In the key first step, the reduction of Y122(•) by the first residue in the hopping pathway, a water ligand to Fe1 of the diferric cluster was suggested to donate a proton to yield the neutral Y122. Here we show that forward radical translocation is associated with perturbation of the Mössbauer spectrum of the diferric cluster, especially the quadrupole doublet associated with Fe1. Density functional theory (DFT) calculations verify the consistency of the experimentally observed perturbation with that expected for deprotonation of the Fe1-coordinated water ligand. The results thus provide the first evidence that the diiron cluster of this prototypical class Ia RNR functions not only in its well-known role as generator of the enzyme's essential Y122(•), but also directly in catalysis.

Full Text

Duke Authors

Cited Authors

  • Wörsdörfer, B; Conner, DA; Yokoyama, K; Livada, J; Seyedsayamdost, M; Jiang, W; Silakov, A; Stubbe, J; Bollinger, JM; Krebs, C

Published Date

  • June 12, 2013

Published In

Volume / Issue

  • 135 / 23

Start / End Page

  • 8585 - 8593

PubMed ID

  • 23676140

Pubmed Central ID

  • 23676140

Electronic International Standard Serial Number (EISSN)

  • 1520-5126

Digital Object Identifier (DOI)

  • 10.1021/ja401342s

Language

  • eng

Conference Location

  • United States