Triple resonance EPR spectroscopy determines the Mn²⁺ coordination to ATP

Mn²⁺ often serves as a paramagnetic substitute to Mg²⁺, providing means for exploring the close environment of Mg²⁺ in many biological systems where it serves as an essential co-factor. This applies to proteins with ATPase activity, where the ATP hydrolysis requires the binding of Mg²⁺-ATP to the ATPase active site. In this context, it is important to distinguish between the Mn²⁺ coordination mode with free ATP in solution as compared to the protein bound case. In this work, we explore the Mn²⁺ complexes with ATP, the non-hydrolysable ATP analog, AMPPNP, and ADP free in solution. Using W-band ³¹P electron-nuclear double resonance (ENDOR) we obtained information about the coordination to the phosphates, whereas from electron-electron double resonance (ELDOR) - detected NMR (EDNMR) we determined the coordination to an adenosine nitrogen. The coordination to these ligands has been reported earlier, but whether the nitrogen and phosphate coordination is within the same nucleotide molecules or different ones is still under debate. By applying the correlation technique, THYCOS (triple hyperfine correlation spectroscopy), and measuring ¹⁵N-³¹P correlations we establish that in Mn-ATP in solution both phosphates and a nitrogen are coordinated to the Mn²⁺ ion. We also carried out DFT calculations to substantiate this finding. In addition, we expanded the understanding of the THYCOS experiment by comparing it to 2D-EDNMR for ⁵⁵Mn-³¹P correlation experiments and through simulations of THYCOS and 2D-EDNMR spectra with ¹⁵N-³¹P correlations.