Characterization of a direct oxygen sensor heme protein from Escherichia coli. Effects of the heme redox states and mutations at the heme-binding site on catalysis and structure.
Characterization of a direct oxygen sensor heme protein from Escherichia coli. Effects of the heme redox states and mutations at the heme-binding site on catalysis and structure.
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DOI：
10.1074/jbc.m202738200
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发表时间：
2002-06
期刊：
影响因子：
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通讯作者：
Yukie Sasakura;S. Hirata;S. Sugiyama;Shingo Suzuki;S. Taguchi;Miki Watanabe;T. Matsui;I. Sagami;Toru Shimizu
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文献类型：
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作者：
Yukie Sasakura;S. Hirata;S. Sugiyama;Shingo Suzuki;S. Taguchi;Miki Watanabe;T. Matsui;I. Sagami;Toru Shimizu
大肠杆菌中含有血红素结合PAS （PAS来自于首次识别不完全重复序列的蛋白名称：PER、ARNT和SIM）结构域的蛋白被认为是一种直接氧传感器（Ec DOS）酶。本研究分离了Ec DOS全长蛋白cDNA，在大肠杆菌中表达，并首次对其结构-功能关系进行了研究。发现Ec - DOS为四聚体，得到6位低自旋铁血红素配合物。CD谱法测定其α -螺旋含量为53%。与SHE相比，血红素的氧化还原电位为+67 mV。分离的PAS结构域His-77的突变使血红素结合消失，而His-83的突变则没有，这表明His-77是血红素轴向配体之一。磷二酯酶（PDE）活性在pH为8.5、Mg（2+）存在的条件下达到最佳，并受到CO、NO和选择性cAMP PDE抑制剂乙唑酸盐的强烈抑制。吸收光谱变化表明CO和NO与亚铁血红素紧密结合。因此，本研究首次明确指出，Ec DOS与cAMP表现出PDE活性，这是由血红素氧化还原状态调节的。
A protein containing a heme-binding PAS (PAS is from the protein names in which imperfect repeat sequences were first recognized: PER, ARNT, and SIM) domain from Escherichia coli has been implied a direct oxygen sensor (Ec DOS) enzyme. In the present study, we isolated cDNA for the Ec DOS full-length protein, expressed it in E. coli, and examined its structure-function relationships for the first time. Ec DOS was found to be tetrameric and was obtained as a 6-coordinate low spin ferric heme complex. Its alpha-helix content was calculated as 53% by CD spectroscopy. The redox potential of the heme was found to be +67 mV versus SHE. Mutation of His-77 of the isolated PAS domain abolished heme binding, whereas mutation of His-83 did not, suggesting that His-77 is one of the heme axial ligands. Ferrous, but not ferric, Ec DOS had phosphodiesterase (PDE) activity of nearly 0.15 min(-1) with cAMP, which was optimal at pH 8.5 in the presence of Mg(2+) and was strongly inhibited by CO, NO, and etazolate, a selective cAMP PDE inhibitor. Absorption spectral changes indicated tight CO and NO bindings to the ferrous heme. Therefore, the present study unequivocally indicates for the first time that Ec DOS exhibits PDE activity with cAMP and that this is regulated by the heme redox state.