Structural basis for ligand discrimination and response initiation in the heme-based oxygen sensor FixL.
Structural basis for ligand discrimination and response initiation in the heme-based oxygen sensor FixL.
复制标题
基于血红素的氧传感器 FixL 中配体辨别和响应启动的结构基础。
DOI：
10.1021/bi9530853
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发表时间：
1996
期刊：
影响因子：
2.9
通讯作者：
J. A. Barron
中科院分区：
文献类型：
--
作者：
K. Rodgers;G. Lukat;J. A. Barron
FixL是一种多结构域细菌O2敏感蛋白，其调节其激酶结构域的活性以响应O2浓度。激酶活性通过磷酰基转移与反应调节蛋白FixJ的转录激活偶联。血红素连接导致从高自旋到低自旋的转变，通过一种迄今尚未明确的机制抑制激酶。本报告提出了光谱，动力学和热力学数据的各种复合物的两个删除衍生物的苜蓿根瘤菌FixL，FixLN（血红素域）和功能血红素激酶，FixL*。metFixLN和metFixL* 的共振拉曼表征表明血红素核心比在高铁肌红蛋白中观察到的小，并且指示metFixL中的五坐标高自旋血红素。FixL-CO加合物的共振拉曼光谱表明，FixL*-CO中的Fe-C = O单元和/或其静电环境相对于FixLN-CO发生了畸变。Met形式的1H NMR光谱进一步支持了与FixL* 中激酶结构域的存在相关的血红素口袋结构的不对称扰动模型。脱氧FixLN和脱氧FixL *（212 cm-1）的等效Fe-咪唑伸缩振动的观察表明，在FixL* 的血红素口袋中的这种扰动的来源不在于血红素的近端侧。deoxyFixLN和FixL* 的等效Fe-咪唑伸缩频率表明，激酶结构域的存在不会改变近端Fe-咪唑键的相对强度，并且近端咪唑配体是弱H-键合的，可能与骨架羰基结合。氰化物和氟离子与FixL反应的动力学和热力学数据与形状选择性一致，这是由于远端血红素口袋中的空间和/或各向异性静电场导致FixL与配体的独特反应性（或缺乏反应性），即，O2、CO、CN-、F-、N3-和SCN-。虽然CN-与metFixLN和metFixL* 结合的速率常数比metMb慢一个数量级，但这些复合物和metMb-CN的稳定性几乎相同。N3-和SCN-都不能以可测量的亲和力与血红素结合。由于其他铁血红素蛋白与这些配体形成稳定的加合物，FixL无法形成类似的复合物表明，这种蛋白质的配体选择性是植根于无法克服的激活障碍的结合含有两个以上的原子的配体和配体的最低能量的协调几何形状是线性的。这使得天然O2配体与其他天然存在的配体在动力学上竞争，这些配体与未受阻碍的血红素形成稳定的复合物。此外，CN-与功能性血红素激酶（metFixL*）结合的速率常数小于其metFixLN对应物，并且metFixL*-CN的稳定性可测量地低于metFixLN-CN的稳定性。这表明FixL* 的血红素和激酶结构域之间的接触对配体结合施加了比FixLN更严格的几何约束。因此，该激酶参与了血红素对配体亲和力的磷酸盐依赖性反馈控制的可能机制。
FixL is a multiple-domain bacterial O2-sensing protein that modulates the activity of its kinase domain in response to O2 concentration. The kinase activity is coupled, via phosphoryl transfer, to transcriptional activation by a response-regulating protein, FixJ. Heme ligation resulting in a transition from high to low spin inhibits the kinase through an, as yet, ill-defined mechanism. This report presents spectroscopic, kinetic, and thermodynamic data on various complexes of two deletion derivatives of Rhizobium meliloti FixL, FixLN (the heme domain) and a functional heme kinase, FixL*. Resonance Raman characterization of metFixLN and metFixL* indicates that the heme core is smaller than that observed in metmyoglobin and is indicative of a five-coordinate high-spin heme in metFixLs. Resonance Raman spectra of FixL-CO adducts reveal that the Fe-C = O unit and/or its electrostatic environment in FixL*-CO is distorted relative to that in FixLN-CO. The 1H NMR spectra of the met forms further support the model of an asymmetric perturbation of the heme pocket structure associated with the presence of the kinase domain in FixL*. Observation of equivalent Fe-imidazole stretching vibrations for deoxyFixLN and deoxyFixL* (212 cm-1) indicates that the source of this perturbation in the heme pocket of FixL* does not lie on the proximal side of the heme. The equivalent Fe-imidazole stretching frequencies for deoxyFixLN and FixL* indicate that the presence of the kinase domain does not alter the relative strength of the proximal Fe-imidazole bond and that the proximal imidazole ligand is weakly H-bonded, probably to a backbone carbonyl group. Kinetic and thermodynamic data for the reactions of cyanide and fluoride ions with FixL are consistent with shape selectivity due to steric and/or an anisotropic electrostatic field in the distal heme pocket being responsible for the unique reactivities (or lack thereof) of FixL with ligands, i.e., O2, CO, CN-, F-, N3-, and SCN-. While the rate constants for binding of CN- to metFixLN and metFixL* are an order of magnitude slower than that for metMb, the stabilities of these complexes and metMb-CN are nearly the same. Neither N3- nor SCN- binds to the heme with measurable affinity. Since other ferric heme proteins form stable adducts with these ligands, the inability of FixL to form analogous complexes suggests that the ligand selectivity of this protein is rooted in insurmountable activation barriers to the binding of ligands containing more than two atoms and for ligands whose lowest-energy coordination geometries are linear. This allows the natural O2 ligand to compete kinetically with other naturally occurring ligands that form stable complexes with unencumbered hemes. Moreover, the rate constant for binding of CN- to the functional heme-kinase (metFixL*) is smaller than its metFixLN counterpart and the stability of metFixL*-CN is measurably lower than that of metFixLN-CN. This indicates that the contacts between the heme and kinase domains of FixL* impose more stringent geometric constraints on ligand binding than FixLN. The kinase is thus implicated in a possible mechanism for phosphate-dependent feedback control over ligand affinity of the heme.