Unconventional Origin of Metal Ion Rescue in the Hammerhead Ribozyme Reaction:  Mn²⁺-Assisted Redox Conversion of 2‘-Mercaptocytidine to Cytidine

Abstract

A specific oxygen atom in RNA is identified as a ligand for a metal ion when sulfur substitution of that atom shifts the metal ion specificity of the RNA-mediated process to a more thiophilic metal. Extensive discussion and debate have centered around whether a metal ion activates the 2′-oxygen nucleophile during the phosphotransesterification reaction catalyzed by the hammerhead ribozyme (HH). To test this possibility, we probed the metal ion specificity of HH reactions using a substrate that contained 2′-mercaptocytidine at the cleavage site. This substrate is generated in situ from a disulfide-protected precursor by treatment with tris(carboxyethyl)phosphine (TCEP). In HH reactions with this substrate, phosphotransesterification does not occur when Mg 2+ is present as the only divalent cation but does occur in the presence of Mn 2+ . These results are consistent with a direct interaction between the metal ion and the nucleophile. However, further analysis reveals that this switch in metal ion specificity does not arise because Mn 2+ coordinates sulfur more readily than Mg 2+ does, but because under the assay conditions, the 2′-mercaptocytidine residue is converted to a mixture of cytidyl-1-β-D-arabinofuranoside and cytidyl-1-β-D-ribofuranoside, the natural substrate for the ribozyme. This conversion occurs in the absence of HH ribozyme, requires Mn 2+ (or Co 2+ ), O 2 , and TCEP, and is inhibited by a free radical scavenger. The mechanism presumably involves a multistep free radical process, in which the key step is homolytic fission of the C2′-sulfur bond induced by TCEP. The putative 2′-carbon radical then reacts with an oxygen species to produce the cytidyl-1-β-D-arabinofuranoside and ribocytidine products. To our knowledge, this chemical transformation is unprecedented in the literature and represents a new reaction for nucleic acids. If O 2 is excluded from the HH reactions, the 2′-sulfur is not modified in the presence of Mn 2+ but is still blocked in the phosphotransesterification reaction, both in the forward direction as the mercapto (-SH) group or in the reverse direction as part of a cyclic phosphorothiolate. Although we are unable to provide evidence for metal ion activation of the nucleophile in the HH ribozyme reaction, this work establishes the groundwork for further use of 2′-mercaptonucleotides in biochemical analyses.

Nucleic acid reactivity: Challenges for next-generation semiempirical quantum models

“…When those molecules are removed from the statistics, both DFTB3‐HNOP and DFTB3 systematically overestimate the proton affinities except the phosphorane set. Chemical modifications that involve the substitution of phosphoryl oxygens with sulfur represent important experimental probes to study metal ion binding and ribozyme mechanisms . Consequently, the modeling of pentavalent phosphorus both with and without thio substitutions is an important area that warrants attention in the development of next‐generation semiempirical quantum models for nucleic acid reactivity.…”

The Origin and Evolution of Ribonucleotide Reduction

“…As a point in case, ribonucleotide reduction has been suggested to have been catalyzed by a ribozyme [ 12 ], but the suggestion has been met with considerable skepticism because of potential problems with free radicals in RNA molecules [ 13 ]. Hsiao et al [ 14 ] have reported redox catalysis by iron in a ribozyme and Hamm et al [ 15 ] have reported that Mn 2+ bound to the Hammerhead ribozyme is more active than Mg 2+ and that activity is affected by radical scavengers. These are indeed intriguing observations, suggesting that RNA may be more resilient to radicals and strong redox metals than hitherto assumed.…”

Tris(2-carboxyethyl)phosphine stabilization of RNA: comparison with dithiothreitol for use with nucleic acid and thiophosphoryl chemistry

“…The mobility of nucleic acids that carry thiophosphate monoesters is markedly diminished (evidence of strong interaction), while thiophosphate diesters showed no evidence of interaction. Disulfides also do not interact with the mercury in the APM gel matrix [19]. Under oxidizing conditions we observe a supershifted band migrating above nonthiophosphorylated RNA, but unretarded by the APM layer, consistent with dimer formation (data not shown).…”

“…When the sulfhydryl-reducing property of TCEP was used to investigate the metal ionbinding properties of the hammerhead ribozyme, this system gave the unexpected result that manganese assisted the redox conversion of 2 0 -mercaptocytidine to cytidine in the presence of TCEP [19]. Conversely, the stability of TCEP itself in the presence of nucleotides has not been previously explored.…”