Department of Biological Chemistry, David Geffen School of Medicine, Molecular Biology Institute, University of California, Los Angeles, California 90095, USA.
J Biol Chem. 2011 May 13;286(19):16716-25. doi: 10.1074/jbc.M110.180844. Epub 2011 Mar 21.
All known heme-thiolate proteins ligate the heme iron using one cysteine side chain. We previously found that DiGeorge Critical Region 8 (DGCR8), an essential microRNA processing factor, associates with heme of unknown redox state when overexpressed in Escherichia coli. On the basis of the similarity of the 450-nm Soret absorption peak of the DGCR8-heme complex to that of cytochrome P450 containing ferrous heme with CO bound, we identified cysteine 352 as a probable axial ligand in DGCR8. Here we further characterize the DGCR8-heme interaction using biochemical and spectroscopic methods. The DGCR8-heme complex is highly stable, with a half-life exceeding 4 days. Mutation of the conserved proline 351 to an alanine increases the rate of heme dissociation and allows the DGCR8-heme complex to be reconstituted biochemically. Surprisingly, DGCR8 binds ferric heme without CO to generate a hyperporphyrin spectrum. The electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance spectra of the DGCR8-heme complex suggest a ferric heme bearing two cysteine ligands. This model was further confirmed using selenomethionine-substituted DGCR8 and mercury titration. DGCR8 is the first example of a heme-binding protein with two endogenous cysteine side chains serving as axial ligands. We further show that native DGCR8 binds heme when expressed in eukaryotic cells. This study provides a chemical basis for understanding the function of the DGCR8-heme interaction in microRNA maturation.
所有已知的含血红素硫醇蛋白质都使用一个半胱氨酸侧链连接血红素铁。我们之前发现，在大肠杆菌中过表达时，DiGeorge 关键区域 8（DGCR8），一种必需的 microRNA 加工因子，与未知氧化还原状态的血红素结合。基于 DGCR8-血红素复合物的 450nm Soret 吸收峰与含有结合 CO 的亚铁血红素的细胞色素 P450 的相似性，我们鉴定出半胱氨酸 352 是 DGCR8 中可能的轴向配体。在这里，我们使用生化和光谱方法进一步表征 DGCR8-血红素相互作用。DGCR8-血红素复合物非常稳定，半衰期超过 4 天。将保守的脯氨酸 351突变为丙氨酸会增加血红素解离的速率，并允许 DGCR8-血红素复合物在生化上重新组成。令人惊讶的是，DGCR8 结合没有 CO 的高铁血红素生成超卟啉光谱。DGCR8-血红素复合物的电子吸收、磁圆二色性和电子顺磁共振光谱表明，高铁血红素带有两个半胱氨酸配体。使用硒代蛋氨酸取代的 DGCR8 和汞滴定进一步证实了该模型。DGCR8 是第一个具有两个内源性半胱氨酸侧链作为轴向配体的血红素结合蛋白的例子。我们进一步表明，在真核细胞中表达时，天然的 DGCR8 结合血红素。这项研究为理解 DGCR8-血红素相互作用在 microRNA 成熟中的功能提供了化学基础。
