Catalysis-dependent selenium incorporation and migration in the nitrogenase active site iron-molybdenum cofactor.
Catalysis-dependent selenium incorporation and migration in the nitrogenase active site iron-molybdenum cofactor.
复制标题
DOI：
10.7554/elife.11620
复制
发表时间：
2015-12-16
期刊：
影响因子：
7.7
通讯作者：
Rees DC
中科院分区：
文献类型：
--
作者：
Spatzal T;Perez KA;Howard JB;Rees DC
固氮酶是一种双组分金属酶，它催化生物氮循环中的二氮还原。了解的惰性静止状态的活性位点FeMo-辅因子转化为活化状态，能够减少二氮仍然难以捉摸。在这里，我们报告的催化依赖性，位点选择性纳入硒的FeMo-辅因子作为一个新确定的底物和抑制剂。1.60 nm分辨率的结构揭示了硒占据棕色固氮菌MoFe蛋白中FeMo辅因子的S2 B位点，该位置最近被鉴定为CO结合位点。Se 2B标记的酶保留底物还原活性，标志着营业额期间的活性位点的晶体学脉冲追踪实验的起点。通过在1.32-1.66 nm的分辨率下获得的一系列晶体结构，包括Av 1-Se 2B的CO抑制形式，证明了所有三个带硫位点的交换，为催化过程中金属中心的不可预见的重排提供了直接的见解。DOI：http://dx.doi.org/10.7554/eLife.11620.001氮元素是所有生命形式所必需的，是重要生物分子如DNA和蛋白质的重要组成部分。氮最丰富的形式是二氮，占地球大气的78%。然而，二氮是高度不反应的，因此氮必须转化为更活泼的形式才能被生物利用。唯一已知的能够进行这种反应的酶被称为固氮酶，但这种酶如何完成这项艰巨的任务仍然不清楚。酶含有一个被称为活性位点的区域，底物-酶作用的分子-与该区域结合。固氮酶的活性部位含有一个称为FeMo-辅因子的区域，该区域必须从非活性状态转变为活性状态以催化二氮转化为氨。固氮酶的另一种底物是一种叫做硒氰酸盐的分子，它由硒原子、碳原子和氮原子组成。Spatzal、Perez等人检查了从棕色固氮菌（Azotobacter vinelandii）中提取的固氮酶的活性位点的结构，同时该酶转化硒代氰酸盐。这揭示了FeMo-辅因子的意外结构变化，这显著挑战了先前关于活性位点如何工作的假设。例如，来自硒氰酸盐的单个硒原子可以掺入FeMo-辅因子的特定位置，这突出了该位置对于酶与底物的初始相互作用的重要性。Spatzal，Perez等人然后使用插入的硒原子作为探针来研究当与称为乙炔的底物反应或被一氧化碳抑制时发生的活性位点结构的变化。这表明硒可以迁移到FeMo辅因子的九个硫原子中的三个（三个“带硫”）在这些相互作用中占据的位置。活性位点以前并没有被认为是以这种方式活跃的：这将需要在所有未来描述二氮如何转化为生物有用形式的模型中考虑。在未来，Spatzal，Perez等人将详细研究这些“带硫”原子如何与来自基底的原子交换，去除的硫储存在哪里，以及它返回的途径。进一步的实验还将表征双氮转化过程中的活性位点。DOI：http://dx.doi.org/10.7554/eLife.11620.002网站
Dinitrogen reduction in the biological nitrogen cycle is catalyzed by nitrogenase, a two-component metalloenzyme. Understanding of the transformation of the inert resting state of the active site FeMo-cofactor into an activated state capable of reducing dinitrogen remains elusive. Here we report the catalysis dependent, site-selective incorporation of selenium into the FeMo-cofactor from selenocyanate as a newly identified substrate and inhibitor. The 1.60 Å resolution structure reveals selenium occupying the S2B site of FeMo-cofactor in the Azotobacter vinelandii MoFe-protein, a position that was recently identified as the CO-binding site. The Se2B-labeled enzyme retains substrate reduction activity and marks the starting point for a crystallographic pulse-chase experiment of the active site during turnover. Through a series of crystal structures obtained at resolutions of 1.32–1.66 Å, including the CO-inhibited form of Av1-Se2B, the exchangeability of all three belt-sulfur sites is demonstrated, providing direct insights into unforeseen rearrangements of the metal center during catalysis. DOI: http://dx.doi.org/10.7554/eLife.11620.001 The element nitrogen is required for all forms of life, and is an essential component of important biological molecules such as DNA and proteins. The most abundant form of nitrogen is dinitrogen, which comprises 78% of the Earth’s atmosphere. However, dinitrogen is highly unreactive, and so the nitrogen must be converted into a more reactive form before it can be used biologically. The only known enzyme capable of carrying out this reaction is called nitrogenase, but how this enzyme performs this difficult task is still not understood. Enzymes contain a region known as the active site, to which substrates – the molecules that the enzyme acts upon – bind. The active site of nitrogenase contains a region called the FeMo-cofactor, which must transform from an inactive to an active state to catalyze the conversion of dinitrogen to ammonia. Another substrate of the nitrogenase enzyme is a molecule called selenocyanate, which is made up of atoms of selenium, carbon and nitrogen. Spatzal, Perez et al. examined the structure of the active site of nitrogenase taken from the bacteria species Azotobacter vinelandii while the enzyme transformed selenocyanate. This revealed unexpected structural changes of the FeMo-cofactor that significantly challenge previous assumptions about how the active site works. For example, a single selenium atom from selenocyanate can be incorporated into a specific position of the FeMo-cofactor, which highlights the importance of this position for the enzyme’s initial interaction with substrates. Spatzal, Perez et al. then used the inserted selenium atom as a probe to investigate the changes in the active site structure that occur when either reacting with a substrate called acetylene or being inhibited by carbon monoxide. This revealed that selenium can migrate into the positions taken up by three of the FeMo-cofactor’s nine sulfur atoms (the three “belt-sulfurs”) during these interactions. The active site was not previously thought to be active in this way: this will need to be taken into account in all future models that describe how dinitrogen is converted into a biologically useful form. In the future, Spatzal, Perez et al. will investigate in detail how these “belt-sulfur” atoms exchange with atoms from the substrate, where the removed sulfur is stored, and the pathway by which it returns. Further experiments will also characterize the active site during the transformation of dinitrogen. DOI: http://dx.doi.org/10.7554/eLife.11620.002