Department of Chemistry and Biochemistry, Auburn University, Auburn, Alabama 36849, USA.
Biochemistry. 2012 Jun 19;51(24):4835-49. doi: 10.1021/bi3001215. Epub 2012 Jun 7.
(E)-4-Hydroxy-3-methylbut-2-enyl diphosphate reductase (IspH or LytB) catalyzes the terminal step of the MEP/DOXP pathway where it converts (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) into the two products, isopentenyl diphosphate and dimethylallyl diphosphate. The reaction involves the reductive elimination of the C4 hydroxyl group, using a total of two electrons. Here we show that the active form of IspH contains a [4Fe-4S] cluster and not the [3Fe-4S] form. Our studies show that the cluster is the direct electron source for the reaction and that a reaction intermediate is bound directly to the cluster. This active form has been trapped in a state, dubbed FeS(A), that was detected by electron paramagnetic resonance (EPR) spectroscopy when one-electron-reduced IspH was incubated with HMBPP. In addition, three mutants of IspH have been prepared and studied, His42, His124, and Glu126 (Aquifex aeolicus numbering), with particular attention paid to the effects on the cluster properties and possible reaction intermediates. None of the mutants significantly affected the properties of the 4Fe-4S cluster, but different effects were observed when one-electron-reduced forms were incubated with HMBPP. Replacing His42 led to an increased K(M) value and a much lower catalytic efficiency, confirming the role of this residue in substrate binding. Replacing the His124 also resulted in a lower catalytic efficiency. In this case, however, the enzyme showed the loss of the 4Fe-4S EPR signal upon addition of HMBPP without the subsequent formation of the FeS(A) signal. Instead, a radical-type signal was observed in some of the samples, indicating that this residue plays a role in the correct positioning of the substrate. The incorrect orientation in the mutant leads to the formation of substrate-based radicals instead of the cluster-bound intermediate complex FeS(A). Replacing the Glu126 also resulted in a lower catalytic efficiency, with yet a third type of EPR signal being detected upon incubation with HMBPP. (31)P and (2)H ENDOR measurements of the FeS(A) species incubated with regular and (2)H-C4-labeled HMBPP reveal that the substrate binds to the enzyme in the proximity of the active-site cluster with C4 adjacent to the site of linkage between the FeS cluster and HMBPP. Comparison of the spectroscopic properties of this intermediate to those of intermediates detected in (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase and ferredoxin:thioredoxin reductase suggests that HMBPP binds to the FeS cluster via its hydroxyl group instead of a side-on binding as previously proposed for the species detected in the inactive Glu126 variant. Consequences for the IspH reaction mechanism are discussed.
（E）-4-羟基-3-甲基-2-丁烯基二磷酸还原酶（IspH 或 LytB）催化 MEP/DOXP 途径的最后一步，将（E）-4-羟基-3-甲基-2-丁烯基二磷酸（HMBPP）转化为两种产物，异戊烯二磷酸和二甲基烯丙基二磷酸。该反应涉及 C4 羟基的还原消除，总共使用两个电子。在这里，我们表明 IspH 的活性形式含有[4Fe-4S]簇，而不是[3Fe-4S]形式。我们的研究表明，该簇是反应的直接电子源，并且反应中间体直接结合到簇上。这种活性形式已被捕获在一种状态中，称为 FeS(A)，当单电子还原的 IspH 与 HMBPP 孵育时，通过电子顺磁共振（EPR）光谱检测到这种状态。此外，已经制备并研究了三种 IspH 的突变体，His42、His124 和 Glu126（Aquifex aeolicus 编号），特别关注对簇性质和可能的反应中间体的影响。这些突变体都没有显著影响4Fe-4S簇的性质，但当单电子还原形式与 HMBPP 孵育时，观察到不同的影响。取代 His42 导致 K(M)值增加和催化效率大大降低，证实了该残基在底物结合中的作用。取代 His124 也导致催化效率降低。然而，在这种情况下，当添加 HMBPP 时，该酶失去了4Fe-4SEPR 信号，而没有随后形成 FeS(A)信号。相反，在一些样品中观察到自由基类型的信号，表明该残基在正确定位底物中起作用。在突变体中不正确的取向导致形成基于底物的自由基而不是簇结合的中间复合物 FeS(A)。取代 Glu126 也导致催化效率降低，并且在用 HMBPP 孵育时还检测到第三种类型的 EPR 信号。用常规和（2）H-C4 标记的 HMBPP 孵育的 FeS(A)物种的（31）P 和（2）H ENDOR 测量表明，底物与酶结合，靠近活性位点簇，C4 与 FeS 簇和 HMBPP 之间的连接位点相邻。将该中间产物的光谱性质与在（E）-4-羟基-3-甲基-2-丁烯基二磷酸合酶和铁氧还蛋白：硫氧还蛋白还原酶中检测到的中间产物的光谱性质进行比较表明，HMBPP 通过其羟基而不是先前提议的用于检测失活 Glu126 变体中的物种的侧位结合方式结合到 FeS 簇上。讨论了对 IspH 反应机制的影响。
