Mn2+ sites in the hammerhead ribozyme investigated by EPR and continuous-wave Q-band ENDOR spectroscopies

Metal ions are critical to the structure and function of many RNA molecules , but measuring detailed coordination environments in RNA is challenging un der solution conditions. The phosphodiester bond cleavage reaction of the h ammerhead ribozyme is activated by Mn2+, which provides a paramagnetic prob e for EPR-based spectroscopic techniques. In this study, EPR and continuous -wave Q-band (34 GHz) electron nuclear double-resonance (ENDOR) spectroscop ies have been used to investigate the coordination environment of a high-af finity Mn2+ site in the hammerhead ribozyme. Small changes in low-temperatu re X-band EPR signals are detected as signatures of Mn2+ ions in the RNA bi nding pocket. H-1 and P-31 Q-band ENDOR spectra are presented for Mn-hammer head and Mn-nucleotide model complexes. The P-31 ENDOR data allow discrimin ation between direct Mn2+-phosphodiester coordination versus coordination t hrough a hydrogen-bonded water molecule. Observation of hyperfine-coupled P -31 (A(P-31) similar to 4 MHz) provides evidence for direct coordination to a phosphodiester group in the hammerhead Mn2+ site. Exchangeable protons f rom aqueous ligands and nonexchangeable protons from base ligands also are examined for the Mn-nucleotide and Mn-ribozyme complexes. These signals ind icate an ordered site for Mn2+ in the hammerhead ribozyme and allow the lig and environment to be predicted, demonstrating the potential of ENDOR spect roscopy as a probe of RNA-metal interactions.