Lanzilotta W N, Fisher K, Seefeldt L C
Department of Chemistry and Biochemistry, Utah State University, Logan 84322, USA.
Biochemistry. 1996 Jun 4;35(22):7188-96. doi: 10.1021/bi9603985.
MgA TP hydrolysis has been proposed to be absolutely required for electron transfer from the nitrogenase iron (Fe) protein to the molybdenum-iron (MoFe) protein. This work presents evidence for primary electron transfer from the Azotobacter vinelandii nitrogenase Fe protein to the MoFe protein in the absence of MgATP hydrolysis. Deletion of an amino acid (Leu 127) in a signal transduction pathway in the Fe protein resulted in an Fe protein conformation resembling the MgATP-bound state. This altered Fe protein (L127delta) was found to bind to the MoFe protein in the absence of MgATP, forming a tight protein complex. Both steady state and stopped-flow transient kinetic measurements suggest that two L127delta Fe proteins bind to one MoFe protein with an extremely high affinity. From pre-steady state kinetic determinations of the rate of complex dissociation, the affinity was found to be at least 350 times tighter than that of the wild-type A. vinelandii nitrogenase complex and at least 20 times tighter than that of the heterologous Clostridium pasteurianum Fe protein-A. vinelandii MoFe protein complex. The L127delta Fe protein-MoFe protein complex was isolated by gel filtration liquid chromatography. Scanning densitometry of an SDS gel of the complex isolated from the gel filtration column revealed a stoichiometry of 1.7 L 127 delta Fe proteins bound per MoFe protein. The L 127 delta Fe protein was found to transfer a single electron from its [4Fe-4S] cluster to the MoFe protein at a rate of 0.2s-1. This compares with the MgATP dependent electron transfer rate of 140 s-1 observed for transfer of an electron from the wild-type Fe protein to the MoFe protein. No substrate reduction (H+ or C2H2) was detected when wild-type MoFe protein was complemented with L 127 delta Fe protein. The MgATP-independent electron transfer from the L 127 delta Fe protein to the MoFe protein required active MoFe protein and was not inhibited by MgADP. EPR spectroscopy of the complex was employed to confirm the electron transfer reaction. These results show that Fe protein in a conformation resembling the MgATP-bound state can transfer at least one electron to the MoFe protein without the need for MgATP hydrolysis.
有人提出，MgATP水解对于从固氮酶铁（Fe）蛋白向钼铁（MoFe）蛋白的电子转移来说是绝对必需的。这项研究提供了证据，表明在没有MgATP水解的情况下，棕色固氮菌固氮酶Fe蛋白能向MoFe蛋白进行初级电子转移。Fe蛋白中信号转导途径上一个氨基酸（Leu 127）的缺失导致Fe蛋白构象类似于结合MgATP的状态。发现这种改变后的Fe蛋白（L127delta）在没有MgATP的情况下能与MoFe蛋白结合，形成紧密的蛋白复合物。稳态和停流瞬态动力学测量均表明，两个L127delta Fe蛋白以极高的亲和力与一个MoFe蛋白结合。通过预稳态动力学测定复合物解离速率，发现其亲和力比野生型棕色固氮菌固氮酶复合物至少高350倍，比异源巴氏梭菌Fe蛋白 - 棕色固氮菌MoFe蛋白复合物至少高20倍。通过凝胶过滤液相色谱法分离出了L127delta Fe蛋白 - MoFe蛋白复合物。对从凝胶过滤柱分离出的复合物进行SDS凝胶扫描光密度测定，结果显示每一个MoFe蛋白结合1.7个L127delta Fe蛋白。发现L127delta Fe蛋白以0.2s-1的速率从其[4Fe-4S]簇向MoFe蛋白转移单个电子。相比之下，观察到从野生型Fe蛋白向MoFe蛋白转移电子时，MgATP依赖的电子转移速率为140 s-1。当野生型MoFe蛋白与L127delta Fe蛋白互补时，未检测到底物还原（H+或C2H2）。从L127delta Fe蛋白到MoFe蛋白的不依赖MgATP的电子转移需要有活性的MoFe蛋白，并且不受MgADP的抑制。利用复合物的电子顺磁共振光谱来证实电子转移反应。这些结果表明，处于类似于结合MgATP状态构象的Fe蛋白能够在不需要MgATP水解的情况下向MoFe蛋白转移至少一个电子。
