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

• Published in: Journal of the American Chemical Society 2009-09, Vol.131 (38), p.13659-13667
• Main Authors: Iwasaki, Toshio, Samoilova, Rimma I, Kounosu, Asako, Ohmori, Daijiro, Dikanov, Sergei A
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Animals; Cysteine - chemistry; Electron Spin Resonance Spectroscopy; Histidine - chemistry; Mitochondrial Proteins - chemistry; Molecular Sequence Data; Protein Structure, Tertiary; Rats
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja903228w

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 Nδ ligand with hyperfine coupling 15 a = 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 Nε 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.