Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Katahira, Aoba-ku, Sendai 980-8577, Japan.
Biochemistry. 2010 Dec 14;49(49):10381-93. doi: 10.1021/bi100733q. Epub 2010 Nov 19.
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, <0.01 min(-1)] of YddV for the first time. Our data indicate that the YddV reaction is the rate-determining step for two consecutive reactions coupled with phosphodiesterase Ec DOS activity on cyclic di-GMP (c-di-GMP) [turnover number of Ec DOS-Fe(II)-O(2), 61 min(-1)]. Thus, O(2) binding and the heme redox switch of YddV appear to be critical factors in the regulation of c-di-GMP homeostasis. The redox potential and autoxidation rate of heme of the isolated heme domain of YddV (YddV-heme) were determined to be -17 mV versus the standard hydrogen electrode and 0.0076 min(-1), respectively. The Fe(II) complexes of Y43A and Y43L mutant proteins (residues at the heme distal side of the isolated heme-bound globin domain of YddV) exhibited very low O(2) affinities, and thus, their Fe(II)-O(2) complexes were not detected on the spectra. The O(2) dissociation rate constant of the Y43W protein was >150 s(-1), which is significantly larger than that of the wild-type 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 ν(Fe-O(2)) (559 cm(-1)) of the Fe(II)-O(2) complex and ν(Fe-CO) (505 cm(-1)) of the Fe(II)-CO complex of Y43F differed from those (ν(Fe-O(2)), 565 cm(-1); ν(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.
从大肠杆菌 (Ec) 中分离出的 YddV 是一种新型球蛋白偶联血红素基氧传感器蛋白，可根据氧可用性响应显示二鸟苷酸环化酶活性。在这项研究中，我们首次定量测定了 YddV 的活性 [Fe(III), 0.066 min(-1); Fe(II)-O(2) 和 Fe(II)-CO, 0.022 min(-1)] [Fe(III), Fe(III)-原卟啉 IX 配合物; Fe(II), Fe(II)-原卟啉 IX 配合物]和非活性形式 [Fe(II)和 Fe(II)-NO, <0.01 min(-1)] 的周转率。我们的数据表明，YddV 反应是两个连续反应的速率决定步骤，与磷酸二酯酶 Ec DOS 对环二鸟苷酸 (c-di-GMP) 的活性偶联 [Ec DOS-Fe(II)-O(2) 的周转率为 61 min(-1)]。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。分离的血红素域 YddV（YddV-血红素）的血红素氧化还原电位和自动氧化速率分别确定为-17 mV 相对于标准氢电极和 0.0076 min(-1)。Y43A 和 Y43L 突变蛋白（位于 YddV 分离血红素结合球蛋白域血红素远端的残基）的 Fe(II) 配合物表现出非常低的 O(2)亲和力，因此，它们的 Fe(II)-O(2) 配合物在光谱中未被检测到。Y43W 蛋白的 O(2)离解速率常数>150 s(-1)，明显大于野生型蛋白 (22 s(-1))。Y43F 和 Y43W 突变蛋白的自动氧化速率常数分别为 0.069 和 0.12 min(-1)，也明显高于野生型蛋白。代表 ν(Fe-O(2)) (559 cm(-1))的 Y43F 和 Y43W 突变蛋白的共振拉曼频率 Fe(II)-O(2) 配合物和 ν(Fe-CO) (505 cm(-1)) Fe(II)-CO 配合物与野生型蛋白的不同（ν(Fe-O(2))，565 cm(-1)；ν(Fe-CO)，495 cm(-1))，表明 Tyr43 与 O(2)和 CO 分子形成氢键。基于这些结果，我们建议位于血红素远端的 Tyr43 对于 O(2)的识别和 Fe(II)-O(2) 配合物的稳定性很重要，因为残基的羟基似乎与 YddV 中与 Fe(II) 配合物结合的 O(2)分子静电相互作用。我们的发现清楚地支持了 Tyr 在氧感应中的作用，因此通过 YddV 和 Ec DOS 催化的 c-di-GMP 从 GTP 到 pGpG 的整体转化率的调节，这可能适用于其他球蛋白偶联的氧传感器酶。
