Connecting nitrogenase intermediates with the kinetic scheme for N2 reduction by a relaxation protocol and identification of the N2 binding state
Connecting nitrogenase intermediates with the kinetic scheme for N2 reduction by a relaxation protocol and identification of the N2 binding state
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DOI：
10.1073/pnas.0610975104
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发表时间：
2007-01-30
影响因子：
11.1
通讯作者：
Hoffman, Brian M.
中科院分区：
文献类型：
--
作者：
Lukoyanov, Dmitriy;Barney, Brett M.;Hoffman, Brian M.
理解固氮酶将N-2还原为NH3的一个主要障碍是不可能将电子传递同步到MoFe蛋白以生成特定的酶中间产物。当中间体在没有同步电子传递的情况下被捕获时，它所积累的电子数n是未知的。因此，中间体不受以n为指标的还原动力学方案的束缚。我们表明，捕获的中间体本身提供了一个“同步准备”的初始态，并且它在253K时的松弛到静止态，阻止电子传递到MoFe蛋白质的条件，可以被分析来揭示n和松弛反应的性质。该方法适用于在有或没有氮气底物的情况下在周转过程中出现的“H+/H-中间体”(A)。A显示至少两个氢化物/质子结合的活性中心铁钼辅助因子(FeMo-CO)的S=1/2电子顺磁共振信号。A向静止态(C)经历两步弛豫：A->B>C，其中B有一个S=3/2 FEMO-co。这两个步骤都显示出很大的溶剂动力学同位素效应：Kie接近3-4(85%D2O)。在N-2还原的Lowe-Thorneley动力学方案的背景下，这些结果提供了强有力的证据，证明在两个弛豫步骤中都形成了H-2，A是催化中心状态，通过n=4个电子的积累被激活以与N-2结合，并且B已经积累了n=2个电子。
A major obstacle to understanding the reduction of N-2 to NH3 by nitrogenase has been the impossibility of synchronizing electron delivery to the MoFe protein for generation of specific enzymatic intermediates. When an intermediate is trapped without synchronous electron delivery, the number of electrons, n, it has accumulated is unknown. Consequently, the intermediate is untethered from kinetic schemes for reduction, which are indexed by n. We show that a trapped intermediate itself provides a "synchronously prepared" initial state, and its relaxation to the resting state at 253 K, conditions that prevent electron delivery to MoFe protein, can be analyzed to reveal n and the nature of the relaxation reactions. The approach is applied to the "H+/H- intermediate" (A) that appears during turnover both in the presence and absence of N2 substrate. A exhibits an S = 1/2 EPR signal from the active-site iron-molybdenum cofactor (FeMo-co) to which are bound at least two hydrides/protons. A undergoes two-step relaxation to the resting state (C): A -> B -> C, where B has an S = 3/2 FeMo-co. Both steps show large solvent kinetic isotope effects: KIE approximate to 3-4 (85% D2O). In the context of the Lowe-Thorneley kinetic scheme for N-2 reduction, these results provide powerful evidence that H-2 is formed in both relaxation steps, that A is the catalytically central state that is activated for N-2 binding by the accumulation of n = 4 electrons, and that B has accumulated n = 2 electrons.