Spectroscopic characterization of Mn and Cd coordination to phosphorothioates in the conserved A9 metal site of the hammerhead ribozyme.

Phosphorothioate modifications have widespread use in the field of nucleic acids. As substitution of sulfur for oxygen can alter metal coordination preferences, the phosphorothioate metal-rescue experiment is a powerful method for identifying metal coordination sites that influence specific properties in a large RNAs. The A9/G10.1 metal binding site of the hammerhead ribozyme (HHRz) has previously been shown to be functionally important through phosphorothioate rescue experiments. While an A9-S substitution is inhibitory in Mg, thiophilic Cd rescues HHRz activity. Mn is also often used in phosphorothioate metal-rescue studies but does not support activity for the A9-S HHRz. Here, we use EPR, electron spin-echo envelope modulation (ESEEM), and X-ray absorption spectroscopic methods to directly probe the structural consequences of Mn and Cd coordination to R and S phosphorothioate modifications at the A9/G10.1 site in the truncated hammerhead ribozyme (tHHRz). The results demonstrate that while Cd does indeed bind to S in the thio-substituted ligand, Mn coordinates to the non‑sulfur oxo group of this phosphorothioate, regardless of isomer. Computational models demonstrate the energetic preference of MnO over MnS coordination in metal-dimethylthiophosphate models. In the case of the tHHRz, the resulting Mn coordination preference of oxygen in either R or S A9 phosphorothioates differentially tunes catalytic activity, with MnO coordination in the A9-S phosphorothioate enzyme being inhibitory.