Transition metal homeostasis is necessary to sustain life. First row transition metals act as cofactors within the cell, performing vital functions ranging from DNA repair to respiration. However, intracellular metal concentrations exceeding physiological requirements may be toxic. In E. coli, the YqjH flavoprotein is thought to play a role in iron homeostasis. YqjH is transcriptionally regulated by the ferric uptake regulator and a newly discovered regulator encoded by yqjI. The apo-form of YqjI is a transcriptional repressor of both the yqjH and yqjI genes. YqjI repressor function is disrupted upon binding of nickel. The YqjI N-terminus is homologous to nickel-binding proteins, implicating this region as a nickel-binding domain. Based on function, yqjI and yqjH should be renamed Ni-responsive Fe-uptake regulator (nfeR) and Ni-responsive Fe-uptake flavoprotein (nfeF), respectively. X-ray Absorption Spectroscopy was employed to characterize the nickel binding site(s) within YqjI. Putative nickel binding ligands were targeted by site-directed mutagenesis and resulting variants were analyzed in vivo for repressor function. Isothermal titration calorimetry and competitive binding assays were used to further quantify nickel interactions with wild-type YqjI and its mutant derivatives. Results indicate plasticity in the nickel binding domain of YqjI. Residues C42 and C43 were found to be required for in vivo response of YqjI to nickel stress, though these residues are not required for in vitro nickel binding. We propose that YqjI may contain a vicinal disulfide bond between C42 and C43 that is important for nickel-responsive allosteric interactions between YqjI domains.

过渡金属的动态平衡对于维持生命是必不可少的。第一排过渡金属充当细胞内的辅助因子，执行从DNA修复到呼吸等重要功能。但是，超过生理要求的细胞内金属浓度可能是有毒的。在大肠杆菌中，YqjH黄素蛋白被认为在铁稳态中起作用。YqjH受铁摄取调节剂和yqjI编码的新发现调节剂的转录调控。YqjI的脱辅基形式是yqjH和yqjI的转录阻遏物基因。YqjI阻遏物功能在镍结合时被破坏。YqjI N末端与镍结合蛋白同源，暗示该区域为镍结合域。根据功能，应将yqjI和yqjH重命名为Ni响应铁摄取调节剂（nfeR）和Ni响应铁摄取黄素蛋白（nfeF）。）， 分别。X射线吸收光谱法用于表征YqjI内的镍结合位点。假定的镍结合配体通过定点诱变靶向，并在体内分析所得变体的阻遏物功能。等温滴定热法和竞争性结合测定法用于进一步定量镍与野生型YqjI及其突变体衍生物的相互作用。结果表明在YqjI的镍结合域中具有可塑性。发现残基C42和C43对于YqjI对镍胁迫的体内应答是必需的，尽管这些残基对于体外镍结合不是必需的。我们建议YqjI可能在C42和C43之间包含一个邻位二硫键，这对YqjI域之间的镍响应性构构相互作用很重要。