Unification of reaction pathway and kinetic scheme for N2 reduction catalyzed by nitrogenase
Unification of reaction pathway and kinetic scheme for N2 reduction catalyzed by nitrogenase
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DOI：
10.1073/pnas.1202197109
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发表时间：
2012-04-10
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;Yang, Zhi-Yong;Hoffman, Brian M.
固氮酶催化 N-2 和质子还原，生成两个 NH3 和一个 H-2。底物结合发生在称为 FeMo 辅因子 (FeMo-co) 的复杂有机金属簇上。每个催化循环都涉及将八个电子/质子顺序传递到该簇，并且该过程已在 Lowe 和 Thorneley 开发的动力学方案中进行了构建。最近，使用二氮烯、甲基二氮烯 (HN=N-CH3) 或肼作为底物，在周转条件下快速冷冻修饰固氮酶可捕获常见的 S = 1/2 中间体，指定为 I。进一步得出结论，N-2 的两个 N 原子交替氢化（“交替”(A) 途径）。在目前的工作中，Q 波段 CW EPR 和 Mo-95 ESEEM 光谱表明，此类样品还包含具有基态“非 Kramer”双峰的整数自旋态 FeMo-co 的常见中间体。该物质被命名为 H，已通过 ESEEM 光谱法使用 N-14、N-15 同位素体加上甲基二氮烯的 H-1、H-2 同位素体的组合进行了表征。结论是：H具有与FeMo-co结合的NH2，对应于N-2加氢的倒数第二个中间体，是积累七个电子/质子并释放出第一个NH3后形成的状态； I 对应于 N-2 还原的最终中间体，即八个电子/质子积累后形成的状态，在释放和再生静息态 FeMo-co 之前，NH3 仍与 FeMo-co 结合。拟议的 Lowe-Thorneley 动力学模型与“快速”交替反应途径的统一代表了固氮酶还原 N-2 的机制草案。
Nitrogenase catalyzes the reduction of N-2 and protons to yield two NH3 and one H-2. Substrate binding occurs at a complex organometallocluster called FeMo-cofactor (FeMo-co). Each catalytic cycle involves the sequential delivery of eight electrons/protons to this cluster, and this process has been framed within a kinetic scheme developed by Lowe and Thorneley. Rapid freezing of a modified nitrogenase under turnover conditions using diazene, methyldiazene (HN=N-CH3), or hydrazine as substrate recently was shown to trap a common S = 1/2 intermediate, designated I. It was further concluded that the two N-atoms of N-2 are hydrogenated alternately ("Alternating" (A) pathway). In the present work, Q-band CW EPR and Mo-95 ESEEM spectroscopy reveal such samples also contain a common intermediate with FeMo-co in an integer-spin state having a ground-state "non-Kramers" doublet. This species, designated H, has been characterized by ESEEM spectroscopy using a combination of N-14,N-15 isotopologs plus H-1,H-2 isotopologs of methyldiazene. It is concluded that: H has NH2 bound to FeMo-co and corresponds to the penultimate intermediate of N-2 hydrogenation, the state formed after the accumulation of seven electrons/protons and the release of the first NH3; I corresponds to the final intermediate in N-2 reduction, the state formed after accumulation of eight electrons/protons, with NH3 still bound to FeMo-co prior to release and regeneration of resting-state FeMo-co. A proposed unification of the Lowe-Thorneley kinetic model with the "prompt" alternating reaction pathway represents a draft mechanism for N-2 reduction by nitrogenase.