Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697, United States.
Biochemistry. 2012 Feb 21;51(7):1518-31. doi: 10.1021/bi2018305. Epub 2012 Feb 8.
The secreted Mycobacterium tuberculosis (Mtb) heme binding protein Rv0203 has been shown to play a role in Mtb heme uptake. In this work, we use spectroscopic (absorption, electron paramagnetic resonance, and magnetic circular dichrosim) methods to further characterize the heme coordination environments of His-tagged and native protein forms, Rv0203-His and Rv0203-notag, respectively. Rv0203-His binds the heme molecule through bis-His coordination and is low-spin in both ferric and ferrous oxidation states. Rv0203-notag is high-spin in both oxidation states and shares spectroscopic similarity with pentacoordinate oxygen-ligated heme proteins. Mutagenesis experiments determined that residues Tyr59, His63, and His89 are required for Rv0203-notag to efficiently bind heme, reinforcing the hypothesis based on our previous structural and mutagenesis studies of Rv0203-His. While Tyr59, His63, and His89 are required for the binding of heme to Rv0203-notag, comparison of the absorption spectra of the Rv0203-notag mutants suggests the heme ligand may be the hydroxyl group of Tyr59, although an exogenous hydroxide cannot be ruled out. Additionally, we measured the heme affinities of Rv0203-His and Rv0203-notag using stopped flow techniques. The rates for binding of heme to Rv0203-His and Rv0203-notag are similar, 115 and 133 μM(-1) s(-1), respectively. However, the heme off rates differ quite dramatically, whereby Rv0203-His gives biphasic dissociation kinetics with fast and slow rates of 0.0019 and 0.0002 s(-1), respectively, and Rv0203-notag has a single off rate of 0.082 s(-1). The spectral and heme binding affinity differences between Rv0203-His and Rv0203-notag suggest that the His tag interferes with heme binding. Furthermore, these results imply that the His tag has the ability to stabilize heme binding as well as alter heme ligand coordination of Rv0203 by providing an unnatural histidine ligand. Moreover, the heme affinity of Rv0203-notag is comparable to that of other heme transport proteins, implying that Rv0203 may act as an extracellular heme transporter.
结核分枝杆菌（Mycobacterium tuberculosis，Mtb）分泌的血红素结合蛋白 Rv0203 已被证明在 Mtb 血红素摄取中发挥作用。在这项工作中，我们使用光谱（吸收、电子顺磁共振和圆二色性）方法进一步表征了 His 标记和天然蛋白形式 Rv0203-His 和 Rv0203-notag 的血红素配位环境。Rv0203-His 通过双 His 配位结合血红素分子，在高铁和亚铁氧化态下均为低自旋。Rv0203-notag 在两种氧化态下均为高自旋，与五配位氧结合的血红素蛋白具有光谱相似性。突变实验确定 Rv0203-notag 中残基 Tyr59、His63 和 His89 对于血红素的有效结合是必需的，这加强了我们之前对 Rv0203-His 的结构和突变研究的假设。虽然 Tyr59、His63 和 His89 对于 Rv0203-notag 与血红素的结合是必需的，但对 Rv0203-notag 突变体的吸收光谱的比较表明，血红素配体可能是 Tyr59 的羟基，尽管不能排除外源性氢氧化物。此外，我们使用停流技术测量了 Rv0203-His 和 Rv0203-notag 的血红素亲和力。血红素与 Rv0203-His 和 Rv0203-notag 的结合速率相似，分别为 115 和 133 μM(-1)s(-1)。然而，血红素的离解速率差异很大，Rv0203-His 给出了双相解离动力学，快速和慢速离解速率分别为 0.0019 和 0.0002 s(-1)，而 Rv0203-notag 只有一个离解速率为 0.082 s(-1)。Rv0203-His 和 Rv0203-notag 之间的光谱和血红素结合亲和力差异表明，His 标签会干扰血红素结合。此外，这些结果表明，His 标签通过提供非天然组氨酸配体，具有稳定血红素结合以及改变 Rv0203 血红素配体配位的能力。此外，Rv0203-notag 的血红素亲和力可与其他血红素转运蛋白相媲美，这意味着 Rv0203 可能作为细胞外血红素转运蛋白发挥作用。
