Important Roles of Tyr43 at the Putative Heme Distal Side in the Oxygen Recognition and Stability of the Fe(II)-O2 Complex of YddV, a Globin-Coupled Heme-Based Oxygen Sensor Diguanylate Cyclase
Important Roles of Tyr43 at the Putative Heme Distal Side in the Oxygen Recognition and Stability of the Fe(II)-O2 Complex of YddV, a Globin-Coupled Heme-Based Oxygen Sensor Diguanylate Cyclase
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DOI：
10.1021/bi100733q
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发表时间：
2010-12-14
期刊：
影响因子：
2.9
通讯作者：
Shimizu, Toru
中科院分区：
文献类型：
--
作者：
Kitanishi, Kenichi;Kobayashi, Kazuo;Shimizu, Toru
来自大肠杆菌(EC)的YddV是一种新型的珠蛋白偶联的基于血红素的氧感受器蛋白，显示出对氧气可获得性做出反应的双鸟苷环化酶活性。在本研究中，我们定量了活性[Fe(III)，0.066分钟(-1)；Fe(II)-O-2和Fe(II)-CO，0.022分钟(-1)][Fe(III)，Fe(III)-原卟啉IX络合物；Fe(II)，Fe(II)-原卟啉IX络合物]和非活性形式[Fe(II)和Fe(II)-NO，150 S(-1)]的周转次数，这明显大于野生型蛋白(22 S(-1))。Y43F和Y43W突变蛋白的自氧化速率常数分别为0.069和0.12min(-1)，也显著高于野生型蛋白。Y43F的Fe(II)-O-2络合物的nu(Fe)_(O2)(559 cm(-1))和Fe(II)-CO络合物的nu(Fe-CO)(505 cm(-1))的共振拉曼光谱与野生型蛋白质的(nu(Fe-O2)，565 cm(-1)；nu(Fe-CO)，495 cm(-1))不同，表明Tyr43与O-2和CO分子都形成了氢键。根据这些结果，我们认为位于血红素远端的Tyr43对于Fe(II)-O-2络合物的O-2识别和稳定性是重要的，因为残基的羟基似乎与YddV中与Fe(II)络合物结合的O-2分子发生了静电相互作用。我们的发现清楚地支持了Tyr在氧感应中的作用，从而通过YddV和EC DOS催化的c-di-GMP调控从GTP到pGpG的整体转换，这可能适用于其他珠蛋白偶联的氧传感器酶。
YddV from Escherichia coli (Ec) is a novel globin-coupled heme-based oxygen sensor protein displaying diguanylate cyclase activity in response to oxygen availability. In this study, we quantified the turnover numbers of the active [Fe(III), 0.066 min(-1); Fe(II)-O-2 and Fe(II)-CO, 0.022 min(-1)] [Fe(III), Fe(III)-protoporphyrin IX complex; Fe(II), Fe(II)-protoporphyrin IX complex] and inactive forms [Fe(II) and Fe(II)-NO, 150 s(-1), which is significantly larger than that of the wildtype protein (22 s(-1)). The autoxidation rate constants of the Y43F and Y43W mutant proteins were 0.069 and 0.12 min(-1), respectively, which are also markedly higher than that of the wild-type protein. The resonance Raman frequencies representing nu(Fe)_(O2) (559 cm(-1)) of the Fe(II)-O-2 complex and nu(Fe-CO) (505 cm(-1)) of the Fe(II)-CO complex of Y43F differed from those (nu(Fe-O2), 565 cm(-1); nu(Fe-CO), 495 cm(-1)) of the wild-type protein, suggesting that Tyr43 forms hydrogen bonds with both O-2 and CO molecules. On the basis of the results, we suggest that Tyr43 located at the heme distal side is important for the O-2 recognition and stability of the Fe(II)-O-2 complex, because the hydroxyl group of the residue appears to interact electrostatically with the O-2 molecule bound to the Fe(II) complex in YddV. Our findings clearly support a role of Tyr in oxygen sensing, and thus modulation of overall conversion from GTP to pGpG via c-di-GMP catalyzed by YddV and Ec DOS, which may be applicable to other globin-coupled oxygen sensor enzymes.