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首页> 外文期刊>Biochemistry >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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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

机译：酪氨酸血红素远端的Tyr43在氧识别和球蛋白偶联的基于血红素的氧传感器双鸟苷酸环化酶YddV的Fe（II）-O2复合物的稳定性中的重要作用。

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YddV from Escherichia coli (Ec) is a novel globin-coupled heme-based oxygen sensor proteinndisplaying diguanylate cyclase activity in response to oxygen availability. In this study, we quantified thenturnover numbers of the active [Fe(III), 0.066 min-1n; Fe(II)-O2 and Fe(II)-CO, 0.022 min-1n] [Fe(III),nFe(III)-protoporphyrin IX complex; Fe(II), Fe(II)-protoporphyrin IX complex] and inactive forms [Fe(II)nand Fe(II)-NO,<0.01min-1n] ofYddVfor the first time.Our data indicate that theYddVreaction is the rate-ndetermining step for two consecutive reactions coupled with phosphodiesterase Ec DOS activity on cyclic di-nGMP (c-di-GMP) [turnover number of EcDOS-Fe(II)-O2, 61min-1n]. Thus,O2 binding and the heme redoxnswitch of YddV appear to be critical factors in the regulation of c-di-GMP homeostasis. The redox potential andnautoxidation rate of heme of the isolated heme domain of YddV (YddV-heme) were determined to be -17 mVnversus the standard hydrogen electrode and 0.0076 min-1n, respectively. The Fe(II) complexes of Y43A andnY43L mutant proteins (residues at the heme distal side of the isolated heme-bound globin domain of YddV)nexhibited very low O2 affinities, and thus, their Fe(II)-O2 complexes were not detected on the spectra. The O2ndissociation rate constant of the Y43Wprotein was>150 sn-1n, which is significantly larger than that of the wild-ntype protein (22 sn-1n). The autoxidation rate constants of the Y43F and Y43Wmutant proteins were 0.069 andn0.12 min-1n, respectively, which are also markedly higher than that of the wild-type protein. The resonancenRaman frequencies representing νFe-O2n(559 cm-1n) of the Fe(II)-O2 complex and νFe-CO (505 cm-1n)ofthenFe(II)-COcomplex ofY43F differed fromthose (νFe-O2n,565cm-1n; νFe-CO,495cm-1n) of thewild-type protein,nsuggesting that Tyr43 forms hydrogen bonds with both O2 and CO molecules. On the basis of the results, wensuggest that Tyr43 located at the heme distal side is important for the O2 recognition and stability of thenFe(II)-O2 complex, because the hydroxyl group of the residue appears to interact electrostatically with the O2nmolecule bound to the Fe(II) complex inYddV.Our findings clearly support a role ofTyr in oxygen sensing, andnthus modulation of overall conversion from GTP to pGpG via c-di-GMP catalyzed by YddV and Ec DOS,nwhich may be applicable to other globin-coupled oxygen sensor enzymes.
机译：来自大肠杆菌（Ec）的YddV是一种新型的球蛋白偶联血红素型氧传感器蛋白，可响应氧的可用性显示出双鸟苷酸环化酶活性。在这项研究中，我们量化了活动的[Fe（III），0.066 min-1n; Fe（II）-O2和Fe（II）-CO，0.022 min-1n] [Fe（III），nFe（III）-原卟啉IX配合物; Fe（II），Fe（II）-原卟啉IX络合物]和非活性形式[Fe（II）n和Fe（II）-NO，<0.01min-1n]首次出现。我们的数据表明，YddV反应速率为-确定两个连续反应的步骤，再加上磷酸二酯酶Ec DOS对环状di-nGMP（c-di-GMP）的活性[EcDOS-Fe（II）-O2的周转数，61min-1n]。因此，O 2结合和YddV的血红素氧化还原开关似乎是调节c-di-GMP动态平衡的关键因素。测得的YddV血红素结构域（YddV-血红素）的血红素的氧化还原电势和钠氧化氧化速率相对于标准氢电极分别为-17 mV和0.0076 min-1n。 Y43A和nY43L突变蛋白的Fe（II）复合物（位于YddV的与血红素结合的球蛋白结构域的血红素远端的残基）抑制了非常低的O2亲和力，因此在它们上未检测到它们的Fe（II）-O2复合物光谱。 Y43W蛋白的O2ndissociociation速率常数大于150 sn-1n，大大大于野生型的22 sn-1n。 Y43F和Y43Wmutant蛋白的自氧化速率常数分别为0.069和n0.12 min-1n，也明显高于野生型蛋白。 Fe（II）-O2络合物的νFe-O2n（559 cm-1n）和Y43F的Fe（II）-CO络合物的νFe-CO（505 cm-1n）的共振拉曼频率不同于（νFe-O2n，565cm-1n; γFe-CO，495cm-1n）的野生型蛋白，暗示Tyr43与O2和CO分子形成氢键。根据结果​​，我们认为位于血红素远端的Tyr43对于Fe（II）-O2络合物的O2识别和稳定性很重要，因为残基的羟基似乎与与H2O2结合的O2分子发生静电相互作用。 YddV中的Fe（II）络合物传感器酶。

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