School of Chemistry, Centre for Molecular and Structural Biochemistry, University of East Anglia, Norwich Research Park, Norwich, UK.
FEBS J. 2011 Jun;278(12):2011-21. doi: 10.1111/j.1742-4658.2011.08117.x. Epub 2011 May 17.
The iron responsive regulator Irr is found in a wide range of α-proteobacteria, where it regulates many genes in response to the essential but toxic metal iron. Unlike Fur, the transcriptional regulator that is used for iron homeostasis by almost all other bacterial lineages, Irr does not sense Fe(2+) directly, but, rather, interacts with a physiologically important form of iron, namely heme. Recent studies of Irr from the N(2)-fixing symbiont Rhizobium leguminosarum (Irr(Rl)) showed that it binds heme with submicromolar affinity at a His-Xxx-His (HxH) motif. This caused the protein to dissociate from its cognate DNA regulatory iron control element box sequences, thus allowing expression of its target genes under iron-replete conditions. In the present study, we report new insights into the mechanisms and consequences of heme binding to Irr. In addition to the HxH motif, Irr binds heme at a second, lower-affinity site. Spectroscopic studies of wild-type Irr and His variants show that His46 and probably His66 are involved in coordinating heme in a low-spin state at this second site. By contrast to the well-studied Irr from Bradyrhizobium japonicum, neither heme site of Irr(Rl) stabilizes ferrous heme. Furthermore, we show that heme-free Irr(Rl) exists as a mixture of dimeric and larger, likely hexameric, forms and that heme binding promotes Irr(Rl) oligomerization. Bioanalytical studies of Irr(Rl) variants showed that this property is not dependent on the HxH motif but is associated with heme binding at the second site.
• Irr binds to irr by molecular sieving (View Interaction 1, 2) • Irr binds to irr by cosedimentation in solution (View interaction).
铁反应调节因子 Irr 存在于广泛的 α-变形菌中，在这些细菌中，它可响应基本但有毒的金属铁来调节许多基因。与几乎所有其他细菌谱系用于铁稳态的转录调节因子 Fur 不同，Irr 不是直接感应 Fe(2+)，而是与一种生理上重要的铁形式，即血红素相互作用。最近对固氮共生菌根瘤菌（Irr(Rl)）中的 Irr 进行的研究表明，它以亚毫摩尔亲和力结合到 His-Xxx-His (HxH) 基序中的血红素。这导致该蛋白与其同源 DNA 调控铁控制元件盒序列解离，从而允许其靶基因在铁充足的条件下表达。在本研究中，我们报告了血红素与 Irr 结合的机制和后果的新见解。除了 HxH 基序外，Irr 还在第二个低亲和力位点结合血红素。对野生型 Irr 和 His 变体的光谱研究表明，His46 可能还有 His66 参与在第二个位点以低自旋状态协调血红素。与研究充分的 Bradyrhizobium japonicum Irr 不同，Irr(Rl)的两个血红素位点都不能稳定亚铁血红素。此外，我们表明无血红素的 Irr(Rl) 以二聚体和更大的、可能是六聚体的形式存在混合物，并且血红素结合促进 Irr(Rl) 寡聚化。对 Irr(Rl)变体的生物分析研究表明，该特性不依赖于 HxH 基序，而是与第二个位点的血红素结合相关。
