Redox reactions of the iron-sulfur cluster in a ribosomal RNA methyltransferase, RumA - Optical and EPR studies
Redox reactions of the iron-sulfur cluster in a ribosomal RNA methyltransferase, RumA - Optical and EPR studies
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DOI：
10.1074/jbc.m405702200
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发表时间：
2004-08-13
影响因子：
4.8
通讯作者：
Gaffney, BJ
中科院分区：
文献类型：
--
作者：
Agarwalla, S;Stroud, RM;Gaffney, BJ
在鲁马中发现了前所未有的[4Fe-4S]铁-硫簇，RumA是使大肠杆菌23 S核糖体RNA中的U1939甲基化的酶（Agarwalla，S.，基利，J.T.，Santi，D.五、和Stroud，R. M.（2002）J.Biol.Chem.277，8835 - 8840; Lee，T. T.，Agarwalla，S.，和Stroud，R. M.（2004）Structure 12，397 - 407）。甲基转移酶反应不涉及氧化还原步骤。为了了解簇在鲁马中的结构和功能作用，我们表征了铁硫簇的氧化还原反应。作为需氧分离的，鲁马在390 nm处表现出可见吸收最大值，并且是EPR沉默的。它不能通过厌氧添加连二亚硫酸盐来还原。通过去氮核黄素/EDTA光还原得到EPR谱，其数量（56%的S = 1/2物种）和细节（g（av）类似于1.96 - 1.93）表明还原的鲁马中存在[4Fe-4S]（1+）簇。铁氰化物对鲁马的氧化导致390 nm谱带的损失以及444和520 nm处出现较低强度谱带。铁氰化物氧化的鲁马的EPR光谱显示了一部分（< 8%）的FeS簇被捕获在[3Fe-4S]（1+）形式（g（av）类似于2.011）中，以及不寻常的自由基样光谱（g'值2.015、2.00和1.95）。鲁马还与一氧化氮反应，产生蛋白质结合的二亚硝酰铁物种的EPR光谱特征。簇的氧化导致其分解，这可能是在细胞中氧化应激条件下调节鲁马活性的机制。序列数据库搜索显示，鲁马同源物广泛存在于各种生命领域，并含有保守且独特的铁硫簇结合基序CX（5）CGGC。
An unprecedented [4Fe-4S] iron-sulfur cluster was found in RumA, the enzyme that methylates U1939 in Escherichia coli 23 S ribosomal RNA ( Agarwalla, S., Kealey, J. T., Santi, D. V., and Stroud, R. M. ( 2002) J. Biol. Chem. 277, 8835 - 8840; Lee, T. T., Agarwalla, S., and Stroud, R. M. ( 2004) Structure 12, 397 - 407). Methyltransferase reactions do not involve a redox step. To understand the structural and functional roles of the cluster in RumA, we have characterized redox reactions of the iron-sulfur cluster. As isolated aerobically, RumA exhibits a visible absorbance maximum at 390 nm and is EPR silent. It cannot be reduced by anaerobic additions of dithionite. Photoreduction by deazariboflavin/EDTA gives EPR spectra, the quantity (56% of S = 1/2 species) and details (g(av) similar to 1.96 - 1.93) of which indicate a [4Fe-4S](1+) cluster in the reduced RumA. Oxidation of RumA by ferricyanide leads to loss of the 390-nm band and appearance of lower intensity bands at 444 and 520 nm. EPR spectra of ferricyanide-oxidized RumA show a fraction (< 8%) of the FeS cluster trapped in the [3Fe-4S](1+) form (g(av) similar to 2.011) together with unusual radical-like spectrum (g' values 2.015, 2.00, and 1.95). RumA also reacts with nitric oxide to give EPR spectra characteristic of the protein-bound iron dinitrosyl species. Oxidation of the cluster leads to its decomposition and that could be a mechanism for regulating the activity of RumA under conditions of oxidative stress in the cell. Sequence data base searches revealed that RumA homologs are widespread in various kingdoms of life and contain a conserved and unique iron-sulfur cluster binding motif, CX(5)CGGC.