Mn²⁺ Sites in the Hammerhead Ribozyme Investigated by EPR and Continuous-Wave Q-band ENDOR Spectroscopies

Abstract

Metal ions are critical to the structure and function of many RNA molecules, but measuring detailed coordination environments in RNA is challenging under solution conditions. The phosphodiester bond cleavage reaction of the hammerhead ribozyme is activated by Mn 2+ , which provides a paramagnetic probe for EPRbased spectroscopic techniques. In this study, EPR and continuous-wave Q-band (34 GHz) electron nuclear double-resonance (ENDOR) spectroscopies have been used to investigate the coordination environment of a high-affinity Mn 2+ site in the hammerhead ribozyme. Small changes in low-temperature X-band EPR signals are detected as signatures of Mn 2+ ions in the RNA binding pocket. 1 H and 31 P Q-band ENDOR spectra are presented for Mn-hammerhead and Mn-nucleotide model complexes. The 31 P ENDOR data allow discrimination between direct Mn 2+ -phosphodiester coordination versus coordination through a hydrogenbonded water molecule. Observation of hyperfine-coupled 31 P (A( 31 P) ∼4 MHz) provides evidence for direct coordination to a phosphodiester group in the hammerhead Mn 2+ site. Exchangeable protons from aqueous ligands and nonexchangeable protons from base ligands also are examined for the Mn-nucleotide and Mnribozyme complexes. These signals indicate an ordered site for Mn 2+ in the hammerhead ribozyme and allow the ligand environment to be predicted, demonstrating the potential of ENDOR spectroscopy as a probe of RNA-metal interactions.

EPR Spectroscopic Characterization of the Manganese Center and a Free Radical in the Oxalate Decarboxylase Reaction

“…1A) is slightly greater than the others immediately downfield. This may be compared with the identical feature seen for Mn(II) bound to the hammerhead ribozyme as contrasted with free Mn(H 2 O) 6 2ϩ , the latter complex showing a more equitable distribution of intensities across this high field hyperfine triplet (26,27). This observation supports protein-bound rather than freely solvated manganous ions as the source of the EPR signals reported herein.…”

“…environment from forbidden {⌬M S ϭ Ϯ1, ⌬M I ϭ Ϯ1} transitions that gain intensity through the small axial zero-field splitting coupling to the hyperfine transition intensity in second and third order perturbation theory (25,26). Notably, the intensity of the higher-field hyperfine line (sharp peak just below 3600 G in the spectrum of Fig.…”

Multifrequency pulsed EPR studies of biologically relevant manganese(II) complexes

“…In comparison with protein-metal binding sites, far less is known about the bonding properties and homogeneity of the metal sites in RNA. Yet as a paramagnetic substitute, Mn(II) allows detailed investigations of these properties in RNA by EPR methods [35].…”

“…Biochemical and EPR spectroscopic studies carried out by DeRose and coworkers [92,94] found that only one of these sites tightly binds its respective metal ion (k d ≤ 10 µM), and that this site must be populated for "catalytic" activity. However, all available spectroscopic evidence indirectly suggested that the high-affinity binding site exists between the N7 of a guanine residue (G10.1) and the phosphate oxygen of an adjacent adenine (A9), about 2.0 nm from the site of RNA cleavage [35,92,[95][96][97][98].Only recently has this tentative assignment been confirmed through ESEEM spectroscopic measurements of site-specific isotope labeling of the G10.1 nitrogens [99]. Herein, we report results from EPR spectroscopic experiments at multiple frequencies to study a set of simple nucleotide and RNA Mn(II) complexes.…”

Exploring Metal Ion Coordination to Nucleic Acids by NMR

“…[37] A further mimic for the diamagnetic Mg 2+ is the paramagnetic Mn 2+ ion. The latter is used for EPR [38,39] and also NMR studies, where the resonances are severely broadened upon binding of Mn 2+ in the vicinity. [26,40,41] Outer sphere binding of Mg 2+ can be probed with [Co(NH 3 ) 6 ] 3+ (see also Section 3).…”