EPR and ⁵⁷Fe ENDOR investigation of 2Fe ferredoxins from Aquifex aeolicus

We have employed EPR and a set of recently developed electron nuclear double resonance (ENDOR) spectroscopies to characterize a suite of [2Fe-2S] ferredoxin clusters from Aquifex aeolicus (Aae Fd1, Fd4, and Fd5). Antiferromagnetic coupling between the Fe^II, S = 2, and Fe ^III, S = 5/2, sites of the [2Fe-2S]⁺ cluster in these proteins creates an S = 1/2 ground state. A complete discussion of the spin-Hamiltonian contributions to g includes new symmetry arguments along with references to related FeS model compounds and their symmetry and EPR properties. Complete ⁵⁷Fe hyperfine coupling (hfc) tensors for each iron, with respective orientations relative to g, have been determined by the use of "stochastic" continuous wave and/or "random hopped" pulsed ENDOR, with the relative utility of the two approaches being emphasized. The reported hyperfine tensors include absolute signs determined by a modified pulsed ENDOR saturation and recovery (PESTRE) technique, RD-PESTRE - a post-processing protocol of the "raw data" that comprises an ENDOR spectrum. The ⁵⁷Fe hyperfine tensor components found by ENDOR are nicely consistent with those previously found by Mössbauer spectroscopy, while accurate tensor orientations are unique to the ENDOR approach. These measurements demonstrate the capabilities of the newly developed methods. The high-precision hfc tensors serve as a benchmark for this class of FeS proteins, while the variation in the ⁵⁷Fe hfc tensors as a function of symmetry in these small FeS clusters provides a reference for higher-nuclearity FeS clusters, such as those found in nitrogenase.