Critical role of the heme axial ligand, Met95, in locking catalysis of the phosphodiesterase from Escherichia coli (Ec DOS) toward cyclic diGMP
Critical role of the heme axial ligand, Met95, in locking catalysis of the phosphodiesterase from Escherichia coli (Ec DOS) toward cyclic diGMP
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DOI：
10.1074/jbc.m701920200
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发表时间：
2007-07-20
影响因子：
4.8
通讯作者：
Shimizu, Toru
中科院分区：
文献类型：
--
作者：
Tanaka, Atsunari;Takahashi, Hiroto;Shimizu, Toru
大肠杆菌血红素调节磷酸二酯酶（Heme-regulated phosphodiesterase from Escherichia coli，Ec DOS）是一种水解环二核苷酸-GMP的气敏酶，它被O-2或CO与亚铁血红素结合激活。与其他已知的血红素调节的气体传感器酶或蛋白质相比，Ec DOS不特异于单个气体配体。由于Arg（97）在血红素远端侧的Ec DOS与O-2分子相互作用和Met（95）作为轴向配体的Fe（II）血红素结合PAS结构域的远端侧的Ec DOS，我们探讨了这些残基的突变对Ec DOS的活性和气体特异性的影响。我们发现，R97 A，R97 I和R97 E突变不显着影响调节磷酸二酯酶活性的Fe（II）-CO和Fe（II）-NO复合物。的Fe（II）-O-2复合物的突变体的磷酸二酯酶活性不能检测到由于快速自氧化和/或低亲和力的O2。相反，即使是无气体的M95 A和M95 L突变体的活性也与气体活化的野生型酶的活性相似。有趣的是，活性的M95 H突变体被部分激活的O-2，CO和NO。光谱分析表明，铁（II）血红素是在5-协调的高自旋状态的M95 A和M95 L突变体，但在M95 H突变体，像野生型Ec DOS，它是在6-协调的低自旋状态。这些结果表明，Met（95）与Fe（II）血红素的配位对于锁定系统至关重要，并且由外部配体的结合或Met（95）处的突变引起的Met（95）周围的全局结构变化释放了催化锁定并激活了催化。
Heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) is a gas-sensor enzyme that hydrolyzes cyclic dinucleotide-GMP, and it is activated by O-2 or CO binding to the Fe(II) heme. In contrast to other well known heme-regulated gas-sensor enzymes or proteins, Ec DOS is not specific for a single gas ligand. Because Arg(97) in the heme distal side in Ec DOS interacts with the O-2 molecule and Met(95) serves as the axial ligand on the distal side of the Fe(II) heme-bound PAS domain of Ec DOS, we explored the effect of mutating these residues on the activity and gas specificity of Ec DOS. We found that R97A, R97I, and R97E mutations do not significantly affect regulation of the phosphodiesterase activities of the Fe(II)-CO and Fe(II)-NO complexes. The phosphodiesterase activities of the Fe(II)-O-2 complexes of the mutants could not be detected due to rapid autoxidation and/or low affinity for O2. In contrast, the activities even of the gas-free M95A and M95L mutants were similar to that of the gas-activated wild-type enzyme. Interestingly, the activity of the M95H mutant was partially activated by O-2, CO, and NO. Spectroscopic analysis indicated that the Fe(II) heme is in the 5-coordinated high-spin state in the M95A and M95L mutants but that in the M95H mutant, like wild-type Ec DOS, it is in the 6-coordinated low-spin state. These results suggest that Met(95) coordination to the Fe(II) heme is critical for locking the system and that global structural changes around Met(95) caused by the binding of the external ligands or mutations at Met(95) releases the catalytic lock and activates catalysis.