Tanaka Atsunari, Takahashi Hiroto, Shimizu Toru
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Katahira, Sendai 980-8577, Japan.
J Biol Chem. 2007 Jul 20;282(29):21301-7. doi: 10.1074/jbc.M701920200. Epub 2007 May 29.
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 O(2). 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.
来自大肠杆菌的血红素调节磷酸二酯酶（Ec DOS）是一种气体传感酶，可水解环二核苷酸 - GMP，它通过氧气（O₂）或一氧化碳（CO）与亚铁血红素（Fe(II)）结合而被激活。与其他知名的血红素调节气体传感酶或蛋白质不同，Ec DOS对单一气体配体不具有特异性。由于Ec DOS血红素远端的精氨酸（Arg(97)）与O₂分子相互作用，且甲硫氨酸（Met(95)）作为Ec DOS中与Fe(II)血红素结合的PAS结构域远端的轴向配体，我们探究了这些残基突变对Ec DOS活性和气体特异性的影响。我们发现，R97A、R97I和R97E突变对Fe(II)-CO和Fe(II)-NO复合物的磷酸二酯酶活性调节没有显著影响。由于快速自氧化和/或对O₂的低亲和力，无法检测到突变体Fe(II)-O₂复合物的磷酸二酯酶活性。相比之下，即使是无气体的M95A和M95L突变体的活性也与气体激活的野生型酶相似。有趣的是，M95H突变体的活性部分被O₂、CO和NO激活。光谱分析表明，M95A和M95L突变体中的Fe(II)血红素处于五配位高自旋状态，但在M95H突变体中，与野生型Ec DOS一样，它处于六配位低自旋状态。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，并且外部配体结合或Met(95)处的突变引起的Met(95)周围的全局结构变化会释放催化锁并激活催化作用。
