The Institute of Scientific and Industrial Research, Osaka University , Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan.
Acc Chem Res. 2017 Jul 18;50(7):1672-1678. doi: 10.1021/acs.accounts.7b00137. Epub 2017 Jun 21.
Bacteria possess molecular biosensors that enable responses to a variety of stressful conditions, including oxidative stress, toxic compounds, and interactions with other organisms, through elaborately coordinated regulation of gene expression. In Escherichia coli and related bacteria, the transcription factor SoxR functions as a sensor of oxidative stress and nitric oxide (NO). SoxR protein contains a [2Fe-2S] cluster essential for its transcription-enhancing activity, which is regulated by redox changes in the [2Fe-2S] cluster. We have explored the mechanistic and structural basis of SoxR proteins function and determined how the chemistry at the [2Fe-2S] cluster causes the subsequent regulatory response. In this Account, I describe our recent achievements in three different areas using physicochemical techniques, primarily pulse radiolysis. First, redox-dependent conformational changes in SoxR-bound DNA were studied by site-specifically replacing selected bases with the fluorescent probes 2-aminopurine and pyrrolocytosine. X-ray analyses of the DNA-SoxR complex in the oxidized state revealed that the DNA structure is distorted in the center regions, resulting in local untwisting of base pairs. However, the inactive, reduced state had remained uncharacterized. We found that reduction of the [2Fe-2S] cluster in the SoxR-DNA complex weakens the fluorescence intensity within a region confined to the central base pairs in the promoter region. Second, the reactions of NO with [2Fe-2S] clusters of E. coli SoxR were analyzed using pulse radiolysis. The transcriptional activation of SoxR in E. coli occurs through direct modification of [2Fe-2S] by NO to form a dinitrosyl iron complex (DNIC). The reaction of NO with [2Fe-2S] cluster of SoxR proceeded nearly quantitatively with concomitant reductive elimination of two equivalents S atoms. Intermediate nitrosylation products, however, were too unstable to observe. We found that the conversion proceeds through at least two steps, with the faster phase being the first reaction of the NO molecule with the [2Fe-2S] cluster. The slower reaction with the second equivalent NO molecule, however, was important for the formation of DNIC. Third, to elucidate the differences between the distinct responses of SoxR proteins from two different species, we studied the interaction of E. coli and Pseudomonas aeruginosa SoxR with superoxide anion using a mutagenic approach. Despite the homology between E. coli SoxR and P. aeruginosa SoxR, the function of P. aeruginosa SoxR differs from that of E. coli. The substitution of E. coli SoxR lysine residues, located close to [2Fe-2S] clusters, into P. aeruginosa SoxR dramatically affected the reaction with superoxide anion.
细菌拥有分子生物传感器，使它们能够对各种应激条件（包括氧化应激、有毒化合物以及与其他生物体的相互作用）做出反应，其机制是通过精心协调的基因表达调控。在大肠杆菌和相关细菌中，转录因子 SoxR 作为氧化应激和一氧化氮 (NO) 的传感器发挥作用。SoxR 蛋白含有一个[2Fe-2S]簇，该簇对于其转录增强活性是必需的，而该活性受[2Fe-2S]簇的氧化还原变化调节。我们使用物理化学技术（主要是脉冲辐射法）探索了 SoxR 蛋白功能的机制和结构基础，并确定了[2Fe-2S]簇的化学性质如何导致随后的调节反应。在本报告中，我将描述我们在三个不同领域的最新研究成果，这些成果主要利用脉冲辐射法取得。首先，通过将荧光探针 2-氨基嘌呤和吡咯胞嘧啶特异性地取代选定的碱基，研究了 SoxR 结合 DNA 的氧化还原依赖性构象变化。在氧化状态下的 DNA-SoxR 复合物的 X 射线分析表明，DNA 结构在中心区域发生扭曲，导致碱基对局部解旋。然而，未表征的是失活的、还原状态。我们发现，SoxR-DNA 复合物中[2Fe-2S]簇的还原会降低启动子区域中心碱基对中受限区域的荧光强度。其次，使用脉冲辐射法分析了大肠杆菌 SoxR 的[2Fe-2S]簇与 NO 的反应。NO 与大肠杆菌 SoxR 的[2Fe-2S]簇反应，通过直接修饰形成二亚硝酰铁配合物 (DNIC)。SoxR 的转录激活是通过 NO 对[2Fe-2S]的直接修饰来实现的。SoxR 与[2Fe-2S]簇的反应几乎定量进行，同时伴随两个等价 S 原子的还原消除。然而，中间硝酰化产物太不稳定而无法观察到。我们发现，该转化至少经过两个步骤，其中较快的步骤是 NO 分子与[2Fe-2S]簇的第一次反应。然而，与第二个当量 NO 分子的较慢反应对于 DNIC 的形成很重要。第三，为了阐明两种不同物种的 SoxR 蛋白的不同反应之间的差异，我们使用诱变方法研究了大肠杆菌和铜绿假单胞菌 SoxR 与超氧阴离子的相互作用。尽管大肠杆菌 SoxR 和铜绿假单胞菌 SoxR 具有同源性，但铜绿假单胞菌 SoxR 的功能与大肠杆菌 SoxR 不同。将大肠杆菌 SoxR 的赖氨酸残基（位于[2Fe-2S]簇附近）替换为铜绿假单胞菌 SoxR，会极大地影响其与超氧阴离子的反应。
