Nitrogenase: a draft mechanism.
Nitrogenase: a draft mechanism.
复制标题
DOI：
10.1021/ar300267m
复制
发表时间：
2013-02-19
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.;Lukoyanov, Dmitriy;Dean, Dennis R.;Seefeldt, Lance C.
生物固氮--将氮气还原为两个NH3分子--养活了超过一半的人类人口。这个反应是由固氮酶催化的，其主要形式，这里讨论，包括一个电子传递铁蛋白和催化钼铁蛋白。固氮酶已被广泛研究，但其催化机理尚不清楚。至少，一个机制必须识别和表征催化过程中形成的每一种中间体，并将这些中间体嵌入到解释它们动态相互转化的动力学框架中。固氮酶的动力学由Lowe-Thorneley(LT)模型描述，该模型为中间体之间的转换提供了速率常数，表示为En，以在MoFe蛋白中积累的电子(和质子)数量n为指标。然而，直到最近，对纯化固氮酶的研究还没有导致EO以外的任何EN状态的表征。在这篇文章中，我们总结了在固氮酶催化过程中形成的三个冷冻捕获中间态的最新表征，以及它们在LT动力学方案中的位置。首先，我们讨论了关键的E4态，它为氮气结合和还原做好了准备，我们称之为“Janus中间体”。在这种状态下，活性中心铁钼辅助因子([7Fe-9S-Mo-C-高柠檬酸]；FeMo-CO)处于静止氧化水平，其四个累积的还原当量以两个[Fe-H-Fe]桥联氢化物的形式储存。另外两个被捕获的中间体含有还原形式的氮气。一种是中间体I，S=1/2 FeMo-co。Endor/HYSCORE测量表明，I，是最终的催化状态E8，NH3产物在其静止的氧化还原水平上与FeMo-CO结合。另一个特征中间体，命名为H，具有整数自旋的FeMo-co(非Kramers；S≥2)。ESEEM测量表明，H与[−NH_2]片段结合，因此对应于E7。这些指定，加上对以前研究的考虑，暗示了一种途径，其中：(I)N2与H2在E4结合，(Ii)N2被迅速还原为N2H2，(Iii)两个N交替氢化形成肼结合的FeMo-CO，以及(Iv)两个NH3在两个进一步的还原步骤中被释放。该方案确定固氮酶遵循“快速交替(P-A)”反应途径，并将催化途径与LT动力学框架相统一。然而，它没有包括固氮酶催化最令人费解的方面之一：氮气结合时强制生成氢，这显然浪费了两个还原当量，从而浪费了ATP水解提供的总能量的25%。在氢化物和氢化物的有机金属化学的背景下，E4将其四个累积的还原当量存储为两个桥式氢化物，这一发现使我们提出了这个谜题的答案。也就是说，在氮气结合时释放氢气包括还原消除两个氢化物以产生与双还原铁结合的氮气。根据P-A方案，两个可用电子和两个活化质子的耦合输送产生了辅因子结合的双氮烯。这一基石完成了固氮酶的机制草案，该机制组织了大量的数据，并打算作为未来实验的基础。
Biological nitrogen fixation — the reduction of N2 to two NH3 molecules — supports more than half the human population. This reaction is catalyzed by the enzyme nitrogenase, whose predominant form, discussed here, comprises an electron-delivery Fe protein and a catalytic MoFe protein. Nitrogenase has been studied extensively but the catalytic mechanism has remained unknown. At minimum, a mechanism must identify and characterize each intermediate formed during catalysis, and embed these intermediates within a kinetic framework that explains their dynamic interconversion. Nitrogenase kinetics have been described by the Lowe-Thorneley (LT) model, which provides rate constants for transformations among intermediates, denoted En, indexed by the number of electrons (and protons), n, that have been accumulated within the MoFe protein. However, until recently, research on purified nitrogenase had not resulted in characterization of any En state beyond Eo. In this article we summarize the recent characterization of three freeze-trapped intermediate states formed during nitrogenase catalysis, and their placement within the LT kinetic scheme. First we discuss the key E4 state, which is primed for N2 binding and reduction and which we refer to as the “Janus intermediate”. This state contains the active-site iron-molybdenum cofactor ([7Fe-9S-Mo-C-homocitrate]; FeMo-co) at its resting oxidation level, its four accumulated reducing equivalents being stored as two [Fe-H-Fe] bridging hydrides. The other two trapped intermediates contain reduced forms of N2. One, intermediate I, has S = 1/2 FeMo-co. ENDOR/HYSCORE measurements indicate that I, is the final catalytic state, E8, having NH3 product bound to FeMo-co at its resting redox level. The other characterized intermediate, designated H, has integer-spin FeMo-co (Non-Kramers; S ≥ 2). ESEEM measurements indicate that H binds the [−NH2] fragment and therefore corresponds to E7. These assignments, plus consideration of previous studies, imply a pathway in which (i) N2 binds at E4 with liberation of H2, (ii) N2 is promptly reduced to N2H2, (iii) the two N’s are hydrogenated alternately to form hydrazine-bound FeMo-co, and (iv) two NH3 are liberated in two further steps of reduction. This proposal identifies nitrogenase as following a ‘Prompt-Alternating (P-A)’ reaction pathway, and unifies the catalytic pathway with the LT kinetic framework. However, it does not incorporate one of the most puzzling aspects of nitrogenase catalysis: obligatory generation of H2 upon N2 binding that apparently ‘wastes’ two reducing equivalents and thus 25% of the total energy supplied by the hydrolysis of ATP. The finding that E4 stores its four accumulated reducing equivalents as two bridging hydrides, considered in the context of the organometallic chemistries of hydrides and dihydrogen, leads us to propose an answer to this puzzle. Namely, that H2 release upon N2 binding involves reductive elimination of two hydrides to yield N2 bound to doubly reduced Fe. Coupled delivery of the two available electrons and two activating protons yields cofactor-bound diazene, in keeping with the P-A scheme. This keystone completes a draft mechanism for nitrogenase that organizes the vast body of data upon which it is formulated, and is intended to serve as a basis for future experiments.
登录
查看更多内容
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：
15
作者：
Kaestner, Johannes;Bloechl, Peter E.
通讯作者：
Bloechl, Peter E.
影响因子：
4.1
作者：
DILWORTH, MJ;EADY, RR
通讯作者：
EADY, RR
DOI：
10.1073/pnas.1202197109
发表时间：
2012-04-10
影响因子：
11.1
作者：
Lukoyanov, Dmitriy;Yang, Zhi-Yong;Hoffman, Brian M.
通讯作者：
Hoffman, Brian M.
影响因子：
3.9
作者：
Dos Santos, Patricia C.;Mayer, Suzanne M.;Dean, Dennis R.
通讯作者：
Dean, Dennis R.