ENDOR/HYSCORE studies of the common intermediate trapped during nitrogenase reduction of N2H2, CH3N2H, and N2H4 support an alternating reaction pathway for N2 reduction.
ENDOR/HYSCORE studies of the common intermediate trapped during nitrogenase reduction of N2H2, CH3N2H, and N2H4 support an alternating reaction pathway for N2 reduction.
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DOI：
10.1021/ja2036018
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发表时间：
2011-08-03
影响因子：
15
通讯作者：
Hoffman, Brian M.
中科院分区：
文献类型：
--
作者：
Lukoyanov, Dmitriy;Dikanov, Sergei A.;Yang, Zhi-Yong;Barney, Brett M.;Samoilova, Rimma I.;Narasimhulu, Kuppala V.;Dean, Dennis R.;Seefeldt, Lance C.;Hoffman, Brian M.
酶促N2还原沿着一个反应途径进行，该反应途径由一系列中间状态组成，这些中间状态是由与固氮酶MoFe蛋白的活性位点铁钼辅因子（FeMo-co）结合的二氮经过六个氢化步骤（e−/H+传递）而产生的。对于反应途径有两种相互竞争的方案，它们涉及不同的中间体。在“远端”（D）途径中，N2中的单个N分三步氢化，直到释放出第一个NH3，然后剩余的氮基-N再氢化三次以产生第二个NH3。在“交替”（A）途径中，两个N替代地交替氢化，在氢化的四个步骤之后形成肼结合的中间体，并且仅在第五个步骤期间释放第一个NH3。最近结合X/Q带EPR和15 N，1，2 H ENDOR测量表明，在以二氮烯或肼为底物的α-70 Ala/α-195 Gln MoFe蛋白质的周转过程中捕获的状态对应于一个共同的中间体（此处表示为I），其中FeMo-co结合底物衍生的[NxHy]部分，本文报道的测量表明，甲基二氮烯的周转产生相同的中间体。在本报告中，我们描述了X/Q带EPR和14/15 N，1，2 H ENDOR/-HYSCORE/ESEEM测量，表征与该部分相关的N原子和质子。实验证实，与N2 H2、CH 3 N2 H和N2 H4的转换实际上产生了共同的中间体I，并表明底物的N-N键在I中被裂解。对这一发现的分析使我们得出结论，固氮酶通过共同的A反应途径还原N2 H2，CH 3 N2 H和N2 H4，并且N2本身也是如此，Fe离子提供反应位点。
Enzymatic N2 reduction proceeds along a reaction pathway comprised of a sequence of intermediate states generated as a dinitrogen bound to the active-site iron-molybdenum cofactor (FeMo-co) of the nitrogenase MoFe protein undergoes six steps of hydrogenation (e−/H+ delivery). There are two competing proposals for the reaction pathway, and they invoke different intermediates. In the ‘Distal’ (D) pathway, a single N of N2 is hydrogenated in three steps until the first NH3 is liberated, then the remaining nitrido-N is hydrogenated three more times to yield the second NH3. In the ‘Alternating’ (A) pathway, the two N’s instead are hydrogenated alternately, with a hydrazine-bound intermediate formed after four steps of hydrogenation and the first NH3 liberated only during the fifth step. A recent combination of X/Q-band EPR and 15N, 1,2H ENDOR measurements suggested that states trapped during turnover of the α-70Ala/α-195Gln MoFe protein with diazene or hydrazine as substrate correspond to a common intermediate (here denoted I) in which FeMo-co binds a substrate-derived [NxHy] moiety, and measurements reported here show that turnover with methyldiazene generates the same intermediate. In the present report we describe X/Q-band EPR and 14/15N, 1,2H ENDOR/-HYSCORE/ESEEM measurements that characterize the N-atom(s) and proton(s) associated with this moiety. The experiments establish that turnover with N2H2, CH3N2H, and N2H4 in fact generates a common intermediate, I, and show that the N-N bond of substrate has been cleaved in I. Analysis of this finding leads us to conclude that nitrogenase reduces N2H2, CH3N2H, and N2H4 via a common A reaction pathway, and that the same is true for N2 itself, with Fe ion(s) providing the site of reaction.
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