Redox-Dependent Dynamics in Heme-Bound Bacterial Iron Response Regulator (Irr) Protein.
Redox-Dependent Dynamics in Heme-Bound Bacterial Iron Response Regulator (Irr) Protein.
复制标题
血红素结合细菌铁反应调节蛋白 (Irr) 的氧化还原依赖性动力学。
DOI：
10.1021/acs.biochem.6b00512
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发表时间：
2016
期刊：
影响因子：
2.9
通讯作者：
Ishimori,Koichiro
中科院分区：
文献类型：
--
作者：
Kobayashi,Kazuo;Nakagaki,Megumi;Ishikawa,Haruto;Iwai,Kazuhiro;O'Brian,MarkR;Ishimori,Koichiro
来自日本缓生根瘤菌的铁反应调节蛋白 (Irr) 介导血红素生物合成的铁依赖性调节。 Irr 会响应血红素可用性而降解，该过程涉及在分子氧存在下血红素与血红素调节基序 (HRM) 中的 Cys-29 结合。在这项工作中，我们通过脉冲放射分解监测瞬时中间体的形成，评估了血红素结合 Irr 的单电子还原动力学。脉冲放射分解产生的水合电子减少了血红素铁结合的 Irr，促进分子氧通过在 420 nm 处具有最大吸收的初始中间体与 Irr 中的血红素铁结合。该初始中间体转化为二级中间体，其最大吸收波长为 425 nm，一阶速率常数为 1.0 × 104s–1。另一方面，Irr 的 Cys-29 → Ala (C29A) 突变体没有经历第二阶段，这意味着在此过程中发生了 Cys-29 与另一个配体的配体交换。血红素结合 Irr 还原过程中的光谱变化表明，CO 与亚铁血红素的结合由两个阶段组成，其值分别为 1.3 × 105 和 2.5 × 104M–1s–1，这一发现与 Irr 中存在两种不同的血红素一致。相比之下，在有氧溶液中，亚铁血红素氧化成三价铁形式被发现是一个两相过程。 C29A 突变体也发生类似的氧化，但这是作为单相过程发生的。我们推测，在氧化过程中会产生蛋白质降解所必需的活性氧。
The iron response regulator (Irr) protein fromBradyrhizobium japonicummediates iron-dependent regulation of heme biosynthesis. Irr degrades in response to heme availability through a process that involves the binding of heme to Cys-29 in the heme regulatory motif (HRM) in the presence of molecular oxygen. In this work, we assessed the dynamics of one-electron reduction of heme-bound Irr by monitoring the formation of transient intermediates by pulse radiolysis. Hydrated electrons generated by pulse radiolysis reduced heme iron-bound Irr, facilitating the binding of molecular oxygen to the heme iron in Irr through an initial intermediate with an absorption maximum at 420 nm. This initial intermediate was converted to a secondary intermediate with an absorption maximum at 425 nm, with a first-order rate constant of 1.0 × 104s–1. The Cys-29 → Ala (C29A) mutant of Irr, on the other hand, did not undergo the secondary phase, implying that ligand exchange of Cys-29 for another ligand takes place during the process. Spectral changes during the reduction of the heme-bound Irr revealed that binding of CO to ferrous heme consisted of two phases withkonvalues of 1.3 × 105and 2.5 × 104M–1s–1, a finding consistent with the presence of two distinct hemes in Irr. In aerobic solutions, by contrast, oxidation of the ferrous heme to the ferric form was found to be a two-phase process. The C29A mutant was similarly oxidized, but this occurred as a single-phase process. We speculate that a reactive oxygen species essential for degradation of the protein is generated during the oxidation process.