Kounosu Asako, Li Zhongrui, Cosper Nathaniel J, Shokes Jacob E, Scott Robert A, Imai Takeo, Urushiyama Akio, Iwasaki Toshio
Department of Biochemistry and Molecular Biology, Nippon Medical School, Sendagi, Bunkyo-ku, Tokyo 113-8602, Japan.
J Biol Chem. 2004 Mar 26;279(13):12519-28. doi: 10.1074/jbc.M305923200. Epub 2004 Jan 15.
We heterologously overproduced a hyperthermostable archaeal low potential (E(m) = -62 mV) Rieske-type ferredoxin (ARF) from Sulfolobus solfataricus strain P-1 and its variants in Escherichia coli to examine the influence of ligand substitutions on the properties of the [2Fe-2S] cluster. While two cysteine ligand residues (Cys(42) and Cys(61)) are essential for the cluster assembly and/or stability, the contributions of the two histidine ligands to the cluster assembly in the archaeal Rieske-type ferredoxin appear to be inequivalent as indicated by much higher stability of the His(64) --> Cys variant (H64C) than the His(44) --> Cys variant (H44C). The x-ray absorption and resonance Raman spectra of the H64C variant firmly established the formation of a novel, oxidized [2Fe-2S] cluster with one histidine and three cysteine ligands in the archaeal Rieske-type protein moiety. Comparative resonance Raman features of the wild-type, natural abundance and uniformly (15)N-labeled ARF and its H64C variant showed significant mixing of the Fe-S and Fe-N stretching characters for an oxidized biological [2Fe-2S] cluster with partial histidine ligation.
我们在大肠杆菌中异源过量表达了来自嗜热栖热菌P-1菌株的一种超嗜热古菌低电位（E(m)= -62 mV） Rieske型铁氧化还原蛋白（ARF）及其变体，以研究配体取代对[2Fe-2S]簇性质的影响。虽然两个半胱氨酸配体残基（Cys(42)和Cys(61)）对于簇的组装和/或稳定性至关重要，但正如His(64)→Cys变体（H64C）比His(44)→Cys变体（H44C）具有更高的稳定性所表明的那样，两个组氨酸配体对古菌Rieske型铁氧化还原蛋白中簇组装的贡献似乎并不等同。H64C变体的X射线吸收和共振拉曼光谱牢固地证实了在古菌Rieske型蛋白质部分中形成了一种新型的、具有一个组氨酸和三个半胱氨酸配体的氧化[2Fe-2S]簇。野生型、天然丰度和均匀（15）N标记的ARF及其H64C变体的比较共振拉曼特征表明，对于具有部分组氨酸连接的氧化生物[2Fe-2S]簇，Fe-S和Fe-N伸缩特征存在显著混合。
