Continuous-wave and pulsed EPR characterization of the [2Fe-2S](Cys)3(His)1 cluster in rat MitoNEET.

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 (15)N-labeled mitoNEET showing one strongly coupled nitrogen from the His87 N(delta) ligand with hyperfine coupling (15)a = 8 MHz. The (14)N and (15)N HYSCORE spectra also exhibit at least two different cross-peaks located near diagonal in the (++) quadrant, with frequencies approximately 2.8 and 2.4 MHz (N2), and the other approximately 4.0 and 3.5 MHz (N1), but did not show any of the larger splitting approximately 1.1-1.4 MHz previously seen with Rieske proteins. Further analysis with partially (15)N(3)-His-labeled protein indicates that His87 N(epsilon) cross-peaks produce resolved features (N2) in the (14)N spectrum but contribute much less than weakly coupled peptide nitrogen species to the (++) quadrant in the (15)N 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.