The effect of spin-lattice relaxation on DEER background decay

The common approach to background removal in double electron-electron resonance (DEER) measurements on frozen solutions with a three-dimensional homogeneous distribution of doubly labeled biomolecules is to fit the background to an exponential decay function. Excluded volume effects or distribution in a dimension lower than three, such as proteins in a membrane, can lead to a stretched exponential decay. In this work, we show that in cases of spin labels with short spin-lattice relaxation time, up to an order of magnitude longer than the DEER trace length, relevant for metal-based spin labels, spin flips that take place during the DEER evolution time affect the background decay shape. This was demonstrated using a series of temperature-dependent DEER measurements on frozen solutions of a nitroxide radical, a Gd(III) complex, Cu(II) ions, and a bis-Gd(III) model complex. As expected, the background decay was exponential for the nitroxide, whereas deviations were noted for Gd(III) and Cu(II). Based on the theoretical approach of Keller et al. (Phys. Chem. Chem. Phys. 21 (2019) 8228-8245), which addresses the effect of spin-lattice relaxation-induced spin flips during the evolution time, we show that the background decay can be fitted to an exponent including a linear and quadratic term in t, which is the position of the pump pulse. Analysis of the data in terms of the probability of spontaneous spin flips induced by spin-lattice relaxation showed that this approach worked well for the high temperature range studied for Gd(III) and Cu(II). At the low temperature range, the spin flips that occured during the DEER evolution time for Gd(III) exceeded the measured spin-lattice relaxation rate and include contributions from spin flips due to another mechanisms, most likely nuclear spin diffusion.