Climbing nitrogenase: toward a mechanism of enzymatic nitrogen fixation.
Climbing nitrogenase: toward a mechanism of enzymatic nitrogen fixation.
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DOI：
10.1021/ar8002128
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发表时间：
2009-05-19
影响因子：
18.3
通讯作者：
Seefeldt, Lance C.
中科院分区：
文献类型：
--
作者：
Hoffman, Brian M.;Dean, Dennis R.;Seefeldt, Lance C.
“固氮”——通过钼依赖性固氮酶将二氮 (N2) 还原为两个氨 (NH3) 分子——对于所有生命都至关重要。尽管经过四十年的研究，固氮酶的机制仍有大量未解之谜，使其成为“酶中的珠穆朗玛峰”。本报告描述了我们通过应对两个相互依赖的挑战来攀登这座山的一个“面”的努力，这两个挑战对于确定生物氮还原机制至关重要。第一个挑战是确定反应途径：与固氮酶的钼铁 (MoFe) 蛋白的催化 FeMoco 因子 (FeMo-co) 结合的每个底物衍生部分的组成和结构。为了克服这一挑战，我们需要区分两类潜在的反应途径：1）“远端”（D）途径，其中H原子在单个N上顺序添加；2）“交替”（A）途径，其中H原子交替添加到N2的两个N原子上。其次，我们需要在公认的 N2 还原 Lowe-Thorneley 动力学方案中表征中间体之间转化的动力学。这个目标要求我们通过实验确定传递到 MoFe 蛋白质的电子/质子数量及其“库存”——驻留在每个反应组分上并以 H2 或 NH3 形式释放的电子/质子的分区。这种“攀登”的主要障碍是无法生成用于表征的 N2 还原中间体。最近，遗传、生化和光谱方法的结合克服了这一障碍。这些实验将活性位点 FeMo 辅因子的四铁 Fe-S 面之一确定为特定的反应位点，表明残基 α70V 的侧链控制对该面的访问，并支持残基 α195H 的侧链参与质子传递。我们现在可以冷冻淬灭捕获 N2 还原途径中间体，并使用 ENDOR/ESEEM 光谱来表征它们。然而，即使成功捕获 N2 还原中间体，也不会同步将电子传递到 MoFe 蛋白质。因此，在 MoFe 形成过程中传递给 MoFe 的电子和质子数量 n 是未知的。为了确定 n 和电子库存，我们最初使用 ENDOR 光谱来分析辅因子内与 FeMo-co 和 57Fe 结合的底物部分。使用该方法的困难促使我们设计了一个强大的动力学协议来确定被捕获的中间体的 n。本报告描述了我们制定的策略，以将固氮酶机制的这一“面貌”纳入我们的视野，并提供其攀登的方法。尽管峰会仍遥遥无期，但我们期待着登顶之路继续取得进展。
“Nitrogen fixation”—the reduction of dinitrogen (N2) to two ammonia (NH3) molecules—by the Mo-dependent nitrogenase is essential for all life. Despite four decades of research, a daunting number of unanswered questions about the mechanism of nitrogenase make it the ‘Everest of enzymes’. This Account describes our efforts to climb one “face” of this mountain by meeting two interdependent challenges central to determining the mechanism of biological N2 reduction. The first challenge is to determine the reaction pathway: the composition and structure of each of the substrate-derived moieties bound to the catalytic FeMocofactor (FeMo-co) of the molybdenum-iron (MoFe) protein of nitrogenase. To overcome this challenge, we need to discriminate between the two classes of potential reaction pathways: 1) a “distal” (D) pathway, in which H atoms add sequentially at a single N or 2) an “alternating” (A) pathway, in which H atoms add alternately to the two N atoms of N2. Secondly, we need to characterize the dynamics of conversion among intermediates within the accepted Lowe-Thorneley kinetic scheme for N2 reduction. That goal requires us to experimentally determine both the number of electrons/protons delivered to the MoFe protein and their “inventory”—a partition into those residing on each of the reaction components and released as H2 or NH3. The principal obstacle to this “climb” has been the inability to generate N2 reduction intermediates for characterization. A combination of genetic, biochemical, and spectroscopic approaches recently overcame this obstacle. These experiments identified one of the four-iron Fe-S faces of the active-site FeMo-cofactor as the specific site of reactivity, indicated that the sidechain of residue α70V controls access to this face, and supported the involvement of the sidechain of residue α195H in proton delivery. We can now freeze-quench trap N2 reduction pathway intermediates and use ENDOR/ESEEM spectroscopies to characterize them. However, even successful trapping of a N2 reduction intermediate occurs without synchronous electron delivery to the MoFe protein. As a result, the number of electrons and protons, n, delivered to MoFe during its formation is unknown. To determine n and the electron inventory, we initially employed ENDOR spectroscopy to analyze the substrate moiety bound to the FeMo-co and 57Fe within the cofactor. Difficulties in using that approach led us to devise a robust kinetic protocol for determining n of a trapped intermediate. This Account describes strategies that we have formulated to bring this “face” of the nitrogenase mechanism into view and afford approaches to its climb. Although the summit remains distant, we look forward to continued progress in the ascent.
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影响因子：
15
作者：
Christie, PD;Lee, HI;Hoffman, BM
通讯作者：
Hoffman, BM
影响因子：
15
作者：
Davydov, R;Matsui, T;Hoffman, BM
通讯作者：
Hoffman, BM
DOI：
10.1073/pnas.0610975104
发表时间：
2007-01-30
影响因子：
11.1
作者：
Lukoyanov, Dmitriy;Barney, Brett M.;Hoffman, Brian M.
通讯作者：
Hoffman, Brian M.
影响因子：
2.9
作者：
Barney, Brett M.;McClead, Jammi;Seefeldt, Lance C.
通讯作者：
Seefeldt, Lance C.
影响因子：
2.9
作者：
Barney, BM;Laryukhin, M;Seefeldt, LC
通讯作者：
Seefeldt, LC