Biophysical and kinetic characterization of HemAT, an aerotaxis receptor from Bacillus subtilis.
Biophysical and kinetic characterization of HemAT, an aerotaxis receptor from Bacillus subtilis.
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DOI：
10.1529/biophysj.104.047936
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发表时间：
2005-04
影响因子：
3.4
通讯作者：
Wei Zhang;J. Olson;G. Phillips
中科院分区：
文献类型：
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作者：
Wei Zhang;J. Olson;G. Phillips
枯草芽孢杆菌的血红素是一种新型的血红素蛋白，负责感知氧气。全长的氯化血红蛋白、感应域(1-178)和Tyr-70突变体的结构和功能特性已经被鉴定。动力学和平衡测量表明，全长的氯化血红素和传感器结构域都显示出两个不同的O(2)结合组分。高亲和力组分具有大约1-2微米的K(离解)和正常的O(2)离解速率常数，k(O2)=50-80 S(-1)。低亲和力组分具有大约50-100微米的K(离解)和大约等于2000 S(-1)的大的O(2)离解速率常数。平衡曲线的低n值和双相特征表明，O(2)与氯化血红素的结合要么与二聚体中的高亲和力亚基和低亲和力亚基独立结合，要么是负协同作用。在传感器结构域中用Phe、Leu或Trp替换Tyr-70(B10)会导致高亲和力和低亲和力成分的k(O2)急剧增加。相反，失去Tyr-70羟基对CO结合的速率和亲和力影响很小。这些结果表明，Tyr-70侧链的高度动力学行为，以及“向上”与“向下”构象的比例强烈地受到铁-配体络合物的性质的影响。由于具有高亲和力和低亲和力的成分，hemat可以在低氧(0-10微米)和好氧(50-250微米)条件下对氧气浓度梯度做出反应，这一特性原则上可能对强大的传感系统非常重要。HEMAT不寻常的配体结合特性表明，不对称性和明显的负协同性在信号转导途径中起着重要作用。
HemAT from Bacillus subtilis is a new type of heme protein responsible for sensing oxygen. The structural and functional properties of the full-length HemAT protein, the sensor domain (1-178), and Tyr-70 mutants have been characterized. Kinetic and equilibrium measurements reveal that both full-length HemAT and the sensor domain show two distinct O(2) binding components. The high-affinity component has a K(dissociation) approximately 1-2 microM and a normal O(2) dissociation rate constant, k(O2) = 50-80 s(-1). The low-affinity component has a K(dissociation) approximately 50-100 microM and a large O(2) dissociation rate constant equal to approximately 2000 s(-1). The low n-value and biphasic character of the equilibrium curve indicate that O(2) binding to HemAT involves either independent binding to high- and low-affinity subunits in the dimer or negative cooperativity. Replacement of Tyr-70(B10) with Phe, Leu, or Trp in the sensor domain causes dramatic increases in k(O2) for both the high- and low-affinity components. In contrast, the rates and affinity for CO binding are little affected by loss of the Tyr-70 hydroxyl group. These results suggest highly dynamic behavior for the Tyr-70 side chain and the fraction of the "up" versus "down" conformation is strongly influenced by the nature of the iron-ligand complex. As a result of having both high- and low-affinity components, HemAT can respond to oxygen concentration gradients under both hypoxic (0-10 microM) and aerobic (50-250 microM) conditions, a property which could, in principle, be important for a robust sensing system. The unusual ligand-binding properties of HemAT suggest that asymmetry and apparent negative cooperativity play an important role in the signal transduction pathway.