Guimarães Wellinson G, Gondim Ana C S, Costa Pedro Mikael da Silva, Gilles-Gonzalez Marie-Alda, Lopes Luiz G F, Carepo Marta S P, Sousa Eduardo H S
Laboratório de Bioinorgânica, Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, CEP 60455-760 Fortaleza, Ceará, Brazil.
Department of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9038, United States.
J Inorg Biochem. 2017 Jul;172:129-137. doi: 10.1016/j.jinorgbio.2017.04.013. Epub 2017 Apr 13.
FixL from Rhizobium etli (ReFixL) is a hybrid oxygen sensor protein. Signal transduction in ReFixL is effected by a switch off of the kinase activity on binding of an oxygen molecule to ferrous heme iron in another domain. Cyanide can also inhibit the kinase activity upon binding to the heme iron in the ferric state. The unfolding by urea of the purified full-length ReFixL in both active pentacoordinate form, met-FixL(Fe) and inactive cyanomet-FixL (Fe-CN) form was monitored by UV-visible absorption spectroscopy, circular dichroism (CD) and fluorescence spectroscopy. The CD and UV-visible absorption spectroscopy revealed two states during unfolding, whereas fluorescence spectroscopy identified a three-state unfolding mechanism. The unfolding mechanism was not altered for the active compared to the inactive state; however, differences in the ΔG were observed. According to the CD results, compared to cyanomet-FixL, met-FixL was more stable towards chemical denaturation by urea (7.2 vs 4.8kJmol). By contrast, electronic spectroscopy monitoring of the Soret band showed cyanomet-FixL to be more stable than met-FixL (18.5 versus 36.2kJmol). For the three-state mechanism exhibited by fluorescence, the ΔG for both denaturation steps were higher for the active-state met-FixL than for cyanomet-FixL. The overall stability of met-FixL is higher in comparison to cyanomet-FixL suggesting a more compact protein in the active form. Nonetheless, hydrogen bonding by bound cyanide in the inactive state promotes the stability of the heme domain. This work supports a model of signal transduction by FixL that is likely shared by other heme-based sensors.
来自墨西哥根瘤菌的FixL（ReFixL）是一种混合氧传感器蛋白。ReFixL中的信号转导是通过氧分子与另一个结构域中的亚铁血红素铁结合时激酶活性的关闭来实现的。氰化物在与铁状态的血红素铁结合时也能抑制激酶活性。通过紫外可见吸收光谱、圆二色性（CD）和荧光光谱监测了纯化的全长ReFixL在活性五配位形式（高铁FixL（Fe））和非活性氰高铁FixL（Fe-CN）形式下被尿素展开的过程。CD和紫外可见吸收光谱显示在展开过程中有两种状态，而荧光光谱确定了一种三态展开机制。与非活性状态相比，活性状态的展开机制没有改变；然而，观察到了ΔG的差异。根据CD结果，与氰高铁FixL相比，高铁FixL对尿素化学变性更稳定（7.2对4.8kJ/mol）。相比之下，对Soret带的电子光谱监测显示氰高铁FixL比高铁FixL更稳定（18.5对36.2kJ/mol）。对于荧光显示的三态机制，活性状态的高铁FixL两个变性步骤的ΔG都高于氰高铁FixL。与氰高铁FixL相比，高铁FixL的整体稳定性更高，这表明活性形式的蛋白质更紧凑。尽管如此，非活性状态下结合的氰化物形成的氢键促进了血红素结构域的稳定性。这项工作支持了FixL信号转导模型，其他基于血红素的传感器可能也有类似模型。
