The N-terminal Domain of Escherichia coli Assimilatory NADPH-Sulfite Reductase Hemoprotein Is an Oligomerization Domain That Mediates Holoenzyme Assembly
The N-terminal Domain of Escherichia coli Assimilatory NADPH-Sulfite Reductase Hemoprotein Is an Oligomerization Domain That Mediates Holoenzyme Assembly
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DOI：
10.1074/jbc.m115.662379
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发表时间：
2015-07-31
影响因子：
4.8
通讯作者：
Stroupe, M. Elizabeth
中科院分区：
文献类型：
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作者：
Askenasy, Isabel;Pennington, Joseph M.;Stroupe, M. Elizabeth
来源于大肠杆菌的同化NADPH亚硫酸盐还原酶（SiR）是一种结构复杂的氧化还原酶，催化亚硫酸盐还原为硫化物。两个亚基，一个是黄素结合黄素蛋白（SiRFP，α亚基），另一个是含铁血红素蛋白（SiRHP，β亚基），组装成约800 kDa的全酶。这两个亚单位如何组装尚不清楚。SiRHP中的富铁辅因子是独特的，因为它们是通过半胱氨酸配体连接到含铁卟啉类（称为siroheme）的Fe 4S 4簇的共价排列。辅因子生物发生和SiR稳定性之间的联系也不明确。通过氢/氘交换和生化分析，我们表明α（8）β（4）SiR全酶通过SiRHP的N末端和SiRFP的NADPH结合结构域组装。通过使用小角X射线散射，我们探讨了SiRHP N-末端寡聚结构域的结构。我们还报告了一种新形式的血红素蛋白，发生在其辅因子的情况下。Apo-SiRHP形成同源四聚体，也依赖于其N末端，其不能与SiRFP组装。从这些结果中，我们提出，同源四聚体的载脂蛋白SiRHP作为一种质量控制机制，以防止形成无活性的全酶的情况下，限制细胞siroheme。
Assimilatory NADPH-sulfite reductase (SiR) from Escherichia coli is a structurally complex oxidoreductase that catalyzes the six-electron reduction of sulfite to sulfide. Two subunits, one a flavin-binding flavoprotein (SiRFP, the alpha subunit) and the other an iron-containing hemoprotein (SiRHP, the beta subunit), assemble to make a holoenzyme of about 800 kDa. How the two subunits assemble is not known. The iron-rich cofactors in SiRHP are unique because they are a covalent arrangement of a Fe4S4 cluster attached through a cysteine ligand to an iron-containing porphyrinoid called siroheme. The link between cofactor biogenesis and SiR stability is also ill-defined. By use of hydrogen/deuterium exchange and biochemical analysis, we show that the alpha(8)beta(4) SiR holoenzyme assembles through the N terminus of SiRHP and the NADPH binding domain of SiRFP. By use of small angle x-ray scattering, we explore the structure of the SiRHP N-terminal oligomerization domain. We also report a novel form of the hemoprotein that occurs in the absence of its cofactors. Apo-SiRHP forms a homotetramer, also dependent on its N terminus, that is unable to assemble with SiRFP. From these results, we propose that homotetramerization of apo-SiRHP serves as a quality control mechanism to prevent formation of inactive holoenzyme in the case of limiting cellular siroheme.