Continuous-Wave and Pulsed EPR Characterization of the [2Fe−2S](Cys)3(His)1 Cluster in Rat MitoNEET

Abstract

CW EPR spectra of reduced [2Fe−2S](Cys)₃(His)₁ clusters of mammalian mitoNEET soluble domain appear to produce features resulting from the interaction of the electron spins of the two adjacent clusters, which can be explained by employing the local spin model. This model favors the reduction of the outermost iron with His87 and Cys83 ligands, which is supported by orientation-selected hyperfine sublevel correlation (HYSCORE) characterization of the uniformly ¹⁵N-labeled mitoNEET showing one strongly coupled nitrogen from the His87 N_δ ligand with hyperfine coupling ¹⁵a = 8 MHz. The ¹⁴N and ¹⁵N HYSCORE spectra also exhibit at least two different cross-peaks located near diagonal in the (++) quadrant, with frequencies ∼2.8 and 2.4 MHz (N2), and the other ∼4.0 and 3.5 MHz (N1), but did not show any of the larger splitting ∼1.1−1.4 MHz previously seen with Rieske proteins. Further analysis with partially ¹⁵N(3)-His-labeled protein indicates that His87 N_ε cross-peaks produce resolved features (N2) in the ¹⁴N spectrum but contribute much less than weakly coupled peptide nitrogen species to the (++) quadrant in the ¹⁵N spectrum. It is suggested that these quantitative data may be used in future functional and theoretical studies on the mammalian mitoNEET [2Fe−2S] cluster system.