Ryle M J, Seefeldt L C
Department of Chemistry and Biochemistry, Utah State University, Logan, 84322, USA.
Biochemistry. 1996 Apr 16;35(15):4766-75. doi: 10.1021/bi960026w.
The present work defines one MgATP signal transduction pathway in the nitrogenase iron (Fe) protein. Deletion of an amino acid (Leu 127) by site-directed mutagenesis in the protein chain between Asp 125, located in the ATP binding site, and Cys 132, a ligand to the [4Fe-4S] cluster, resulted in protein conformational changes resembling the MgATP-bound state in the absence of any bound nucleotides. Specifically, 1H nuclear magnetic resonance, electron paramagnetic resonance, and circular dichroism spectroscopic properties, along with Fe chelation assays, suggested that deletion of Leu 127 in the Fe protein resulted in changes in the electronic properties of the [4Fe-4S] cluster similar to those normally observed upon MgATP binding to the wild-type Fe protein. Deletion of Leu 127 of the Fe protein lowered the redox potential of of the [4FE-4S] cluster by 112 mV compared to the wild-typeFe protein (-412mV compared to -294 mV). A nearly identical lowering of the redox potential by 120 mV occurs in the wild-type Fe protein upon binding MgATP (-294 mV compared to 420 mV). The L127delta Fe protein did not contain bound nucleotides which could account for the observed conformational changes. The present results support a model in which the protein chain from ASP 125 to Cys 132 acts as one pathway for MgATP signal transduction and suggests a mechanism for this transduction to the [4Fe-4S] cluster. The L127delta Fe protein was found to still bind 2 MgATP or 2 MgADP molecules/Fe protein. Unlike the wild-type Fe protein, the L127delta Fe protein bound 2 ADP molecules/Fe protein in the absence of Mg2+. Finally, the L127delta protein was found to bind to the MoFe protein, although the complex did not catalyze MgATP hydrolysis or substrate reduction. In concurrence with previous models, homologies between the Asp 125 to Cys 132 transduction pathway in Fe protein and the switch II region of the broad class of GTPase signal transduction proteins (G-proteins) are discussed.
本研究确定了固氮酶铁（Fe）蛋白中的一条MgATP信号转导途径。通过定点诱变在位于ATP结合位点的Asp 125与作为[4Fe-4S]簇配体的Cys 132之间的蛋白质链中缺失一个氨基酸（Leu 127），导致蛋白质构象变化，类似于在不存在任何结合核苷酸的情况下MgATP结合状态。具体而言，1H核磁共振、电子顺磁共振和圆二色光谱性质，以及铁螯合测定表明，Fe蛋白中Leu 127的缺失导致[4Fe-4S]簇的电子性质发生变化，类似于通常在MgATP与野生型Fe蛋白结合时观察到的变化。与野生型Fe蛋白相比，Fe蛋白Leu 127的缺失使[4FE-4S]簇的氧化还原电位降低了112 mV（与-294 mV相比为-412 mV）。野生型Fe蛋白在结合MgATP时氧化还原电位也会降低120 mV（与420 mV相比为-294 mV），二者几乎相同。L127delta Fe蛋白不含有可解释观察到的构象变化的结合核苷酸。目前的结果支持一个模型，即从Asp 125到Cys 132的蛋白质链作为MgATP信号转导的一条途径，并提出了这种转导至[4Fe-4S]簇的机制。发现L127delta Fe蛋白仍能结合2个MgATP或2个MgADP分子/Fe蛋白。与野生型Fe蛋白不同，L127delta Fe蛋白在不存在Mg2+的情况下能结合2个ADP分子/Fe蛋白。最后，发现L127delta蛋白能与MoFe蛋白结合，尽管该复合物不催化MgATP水解或底物还原。与之前的模型一致，讨论了Fe蛋白中Asp 125到Cys 132转导途径与广泛的GTPase信号转导蛋白（G蛋白）的开关II区域之间的同源性。
