NifA is a σ⁵⁴ activator that turns on bacterial nitrogen fixation under reducing conditions and when fixed cellular nitrogen levels are low. The redox sensing mechanism in NifA is poorly understood. In α- and β-proteobacteria, redox sensing involves two pairs of Cys residues within and immediately following the protein’s central AAA⁺ domain. In this work, we examine if an additional Cys pair that is part of a C(X)₅ C motif and located immediately upstream of the DNA binding domain of NifA from the α-proteobacterium Gluconacetobacter diazotrophicus (Gd) is involved in redox sensing. We hypothesize that the Cys residues’ redox state may directly influence the DNA binding domain’s DNA binding affinity and/or alter the protein’s oligomeric sate. Two DNA binding domain constructs were generated, a longer construct (2C-DBD), consisting of the DNA binding domain with the upstream Cys pair, and a shorter construct (NC-DBD) that lacks the Cys pair. The K_d of NC-DBD for its cognate DNA sequence (nifH-UAS) is equal to 20.0 µM. The K_d of 2C-DBD for nifH-UAS when the Cys pair is oxidized is 34.5 µM. Reduction of the disulfide bond does not change the DNA binding affinity. Additional experiments indicate that the redox state of the Cys residues does not influence the secondary structure or oligomerization state of the NifA DNA binding domain. Together, these results demonstrate that the Cys pair upstream of the DNA binding domain of Gd-NifA does not regulate DNA binding or domain dimerization in a redox dependent manner.

NifA 是一种 σ ⁵⁴激活剂，可在还原条件下以及细胞固定氮水平较低时启动细菌固氮。NifA 中的氧化还原传感机制尚不清楚。在 α- 和 β- 变形菌中，氧化还原传感涉及蛋白质中央 AAA ⁺结构域内并紧随其后的两对 Cys 残基。在这项工作中，我们检查了作为 C(X) ₅  C 基序一部分且位于来自 α-变形菌固氮葡糖酸杆菌( Gd ) 的 NifA DNA 结合域紧邻上游的额外 Cys 对是否参与氧化还原传感。我们假设Cys残基的氧化还原状态可能直接影响DNA结合结构域的DNA结合亲和力和/或改变蛋白质的寡聚状态。生成了两个 DNA 结合结构域构建体，一个较长的构建体 (2C-DBD)，由具有上游 Cys 对的 DNA 结合结构域组成，另一个较短的构建体 (NC-DBD) 缺乏 Cys 对。NC -DBD 的同源 DNA 序列 (nifH-UAS) 的 K d_等于20.0 µM。当 Cys 对被氧化时，nifH-UAS 的 2C-DBD 的K d_{为34.5 µM。}二硫键的还原不会改变 DNA 结合亲和力。其他实验表明 Cys 残基的氧化还原状态不会影响 NifA DNA 结合结构域的二级结构或寡聚状态。总之，这些结果证明Gd -NifA的DNA结合结构域上游的Cys对不以氧化还原依赖性方式调节DNA结合或结构域二聚化。