Combining steady-state and dynamic methods for determining absolute signs of hyperfine interactions: Pulsed ENDOR Saturation and Recovery (PESTRE)

The underlying causes of asymmetric intensities in Davies pulsed ENDOR spectra that are associated with the signs of the hyperfine interaction are reinvestigated. The intensity variations in these asymmetric ENDOR patterns are best described as shifts in an apparent baseline intensity that occurs dynamically following on-resonance ENDOR transitions. We have developed an extremely straightforward multi-sequence protocol that is capable of giving the sign of the hyperfine interaction by probing a single ENDOR transition, without reference to its partner transition. This technique, Pulsed ENDOR Saturation and Recovery (PESTRE) monitors dynamic shifts in the 'baseline' following measurements at a single RF frequency (single ENDOR peak), rather than observing anomalous ENDOR intensity differences between the two branches of an ENDOR response. These baseline shifts, referred to as dynamic reference levels (DRLs), can be directly tied to the electron-spin manifold from which that ENDOR transition arises. The application of this protocol is demonstrated on ⁵⁷Fe ENDOR of a 2Fe-2S ferredoxin. We use the ¹⁴N ENDOR transitions of the S = 3/2[Fe(II)NO]²⁺ center of the non-heme iron enzyme, anthranilate dioxygenase (AntDO) to examine the details of the relaxation model using PESTRE.