Characterizing multiple metal ion binding sites within a ribozyme by Cadmium-induced EPR silencing

N. Kisseleva, S. Kraut, A. Jäschke, Olav Schiemann

Research output: Contribution to journalArticlepeer-review

28 Citations (Scopus)

Abstract

In ribozyme catalysis, metal ions are generally known to make structural and/or mechanistic contributions. The catalytic activity of a previously described Diels-Alderase ribozyme was found to depend on the concentration of divalent metal ions, and crystallographic data revealed multiple binding sites. Here, we elucidate the interactions of this ribozyme with divalent metal ions in solution using electron paramagnetic resonance, EPR. spectroscopy. Manganese ion titrations revealed five high-affinity Mn2+ binding sites with an upper K-d of 0.6 +/- 0.2 mu M. In order to characterize each binding site individually, EPR-silent Cd2+ ions were used to saturate the other binding sites. This cadmium-induced EPR silencing showed that the Mn2+ binding sites possess different affinities. In addition, these binding sites could be assigned to three different types, including innersphere, outersphere, and a Mn2+ dimer. Based on simulations, the Mn2+-Mn2+ distance within the dimer was found to be similar to 6 angstrom, which is in good agreement with crystallographic data. The EPR-spectroscopic characterization reveals no structural changes upon addition of a Diels-Alder product, supporting the concept of a preorganized catalytic pocket in the Diels-Alder ribozyme and the structural role of these ions.

Original languageEnglish
Pages (from-to)127-136
Number of pages10
JournalHFSP Journal
Volume1
Issue number2
DOIs
Publication statusPublished - Jul 2007

Keywords

  • ELECTRON-PARAMAGNETIC-RESONANCE
  • DIELS-ALDERASE RIBOZYME
  • HAMMERHEAD RIBOZYME
  • ACTIVE-SITE
  • LIVER ARGINASE
  • DIMANGANESE(II,II) CENTER
  • CATALYSIS
  • MANGANESE(II)
  • SPECTROSCOPY
  • COMPLEXES

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