Diazene (HN=NH) is a substrate for nitrogenase:: Insights into the pathway of N2 reduction
Diazene (HN=NH) is a substrate for nitrogenase:: Insights into the pathway of N2 reduction
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DOI：
10.1021/bi062294s
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发表时间：
2007-06-12
期刊：
影响因子：
2.9
通讯作者：
Seefeldt, Lance C.
中科院分区：
文献类型：
--
作者：
Barney, Brett M.;McClead, Jammi;Seefeldt, Lance C.
固氮酶催化六个电子和六个质子顺序添加到与活性位点金属簇FeMo-辅因子结合的N-2，产生两个氨分子。沿着沿着该还原途径与FeMo-辅因子结合的中间体的性质仍然未知，尽管已经提出在二氮烯（HN=NH，也称为二酰亚胺）和肼（H2 N-NH 2）的还原水平存在中间体。通过在固氮酶周转过程中原位生成的二氮烯，我们表明，二氮烯是野生型固氮酶的底物，并被还原为NH3。二氮烯还原，如N-2还原，被H-2抑制。这与固氮酶还原肼时没有H-2抑制形成对比。这些结果支持在N-2还原途径的早期存在一个中间体，在还原二氮烯的水平上。冷冻淬灭MoFe蛋白变体，在用二氮烯进行稳态转换期间，α-195（His）被Gln取代和α-70（瓦尔）被Ala取代导致S = 3 /2静息状态FeMo-辅因子转化为新的S = 1/2状态，g（1）= 2.09，g（2）= 2.01，和g（3）类似于1-98 N-15-和H-1-ENDOR，确定了这种状态由与FeMo-辅因子结合的二氮烯衍生的[-NHx]部分组成。该部分是无法区分的肼衍生的[-NHx]部分绑定到FeMo-辅因子时，相同的MoFe蛋白被困在营业额与肼。这些观察结果表明，二氮烯加入正常的N-2-还原途径，二氮烯和肼捕获的周转状态代表相同的中间体在正常的N-2还原固氮酶。这些发现的影响固氮酶的N-2还原的机制进行了讨论。
Nitrogenase catalyzes the sequential addition of six electrons and six protons to a N-2 that is bound to the active site metal cluster FeMo-cofactor, yielding two ammonia molecules. The nature of the intermediates bound to FeMo-cofactor along this reduction pathway remains unknown, although it has been suggested that there are intermediates at the level of reduction of diazene (HN=NH, also called diimide) and hydrazine (H2N-NH2). Through in situ generation of diazene during nitrogenase turnover, we show that diazene is a substrate for the wild-type nitrogenase and is reduced to NH3. Diazene reduction, like N-2 reduction, is inhibited by H-2. This contrasts with the absence of H-2 inhibition when nitrogenase reduces hydrazine. These results support the existence of an intermediate early in the N-2 reduction pathway at the level of reduction of diazene. Freeze-quenching a MoFe protein variant with (alpha-195(His) substituted by Gln and alpha-70(Val) substituted by Ala during steady-state turnover with diazene resulted in conversion of the S = 3 /2 resting state FeMo-cofactor to a novel S = 1/2 state with g(1) = 2.09, g(2) = 2.01, and g(3) similar to 1-98 N-15- and H-1-ENDOR establish that this state consists of a diazene-derived [-NHx] moiety bound to FeMo-cofactor. This moiety is indistinguishable from the hydrazine-derived [-NHx] moiety bound to FeMo-cofactor when the same MoFe protein is trapped during turnover with hydrazine. These observations suggest that diazene joins the normal N-2-reduction pathway, and that the diazene- and hydrazine-trapped turnover states represent the same intermediate in the normal reduction of N-2 by nitrogenase. Implications of these findings for the mechanism of N-2 reduction by nitrogenase are discussed.