Testing if the interstitial atom, X, of the nitrogenase molybdenum-iron cofactor is N or C: ENDOR, ESEEM, and DFT studies of the S=3/2 resting state in multiple environments
Testing if the interstitial atom, X, of the nitrogenase molybdenum-iron cofactor is N or C: ENDOR, ESEEM, and DFT studies of the S=3/2 resting state in multiple environments
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DOI：
10.1021/ic7018814
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发表时间：
2007-12-24
影响因子：
4.6
通讯作者：
Hoffman, Brian M.
中科院分区：
文献类型：
--
作者：
Lukoyanov, Dmitriy;Pelmenschikov, Vladimir;Hoffman, Brian M.
固氮酶铁（MoFe）蛋白质的高分辨率（1.16 A）X射线结构揭示了[MoFe 7S 9：高柠檬酸盐] FeMo-辅因子（FeMo-co）中心铁棱柱（[6 Fe]）内单个N、O或C原子（表示为X）的电子密度。在此，我们扩展了早期的努力，通过在三种环境中对FeMo-co的N-14/15和C-12/13同位素异构体进行互锁和相互支持的9 GHz电子自旋回波包络调制（ESEEM）和85 GHz电子-核双共振（ENDOR）测量，通过详细测试X = N还是C来确定X的身份：（i）掺入天然MoFe蛋白质环境;（ii）提取到N-甲基甲酰胺溶液中;和（iii）掺入到NifX蛋白中，其在FeMo-co生物合成期间充当伴侣。这些测量提供了强有力的证据，X; N/C，除非X实际上与静止FeMo-co的S = 3/2电子自旋系统磁解耦。它们显示在三种环境中的任何一种中都没有来自FeMo-co的信号，可以从N-14/15或C-13分配给X：如果X是任一种元素，则在所有测量场处观察到的最大超精细耦合估计为A（（14/15）Nx）< 0.07/0.1 MHz，A（（13）Cx）< 0.1 MHz，对应于这些值的大约一半的固有耦合。同时，我们用密度泛函理论（DFT）结合自旋耦合的对称破缺（BS）方法计算了X = N-14/15/C-13/O-17的超精细张量，X = N-14的核四极耦合常数e（2）qQ，以及S = 3/2FeMo-co的Fe位的超精细常数.当X = C/N时，实验所要求的退耦强有力地支持FeMo-Co铁位的“BS 7”自旋耦合，其中小的X超精细耦合是三个自旋向上和三个自旋向下的自旋密度贡献精确平衡的结果（3个向上箭头：3个向下箭头）FeMo-co的[6 Fe] prismane“腰部”的铁原子;这将排除“BS 6”分配（4个向上箭头：2个向下箭头表示[6 Fe]）。然而，即使使用BS 7方案，在g（2）附近观察到的X的超精细耦合也足够大，它们应该已经被检测到：我们认为，只有当a（iso）03 -0.07/0.05-0.1 MHz和ai（so）（C-13（X））<0.05-0.1 MHz，与aiso（N-14/15（X））= 0.3/0.4 MHz和a（iso）（C-13（X））= 1 MHz的计算值相比。然而，DFT的不确定性足够大，实验所需的非常小的超精细耦合并不一定排除X = N/C。
A high-resolution (1.16 A) X-ray structure of the nitrogenase molybdenum-iron (MoFe) protein revealed electron density from a single N, O, or C atom (denoted X) inside the central iron prismane ([6Fe]) of the [MoFe7S9: homocitrate] FeMo-cofactor (FeMo-co). We here extend earlier efforts to determine the identity of X through detailed tests of whether X = N or C by interlocking and mutually supportive 9 GHz electron spin echo envelope modulation (ESEEM) and 85 GHz electron-nuclear double resonance (ENDOR) measurements on N-14/15 and C-12/13 isotopomers of FeMo-co in three environments: (i) incorporated into the native MoFe protein environment; (ii) extracted into N-methyl formamide solution; and (iii) incorporated into the NifX protein, which acts as a chaperone during FeMo-co biosynthesis. These measurements provide powerful evidence that X; N/C, unless X in effect is magnetically decoupled from the S = 3/2 electron spin system of resting FeMo-co. They reveal no signals from FeMo-co in any of the three environments that can be assigned to X from either N-14/15 or C-13: If X were either element, its maximum observed hyperfine coupling at all fields of measurement is estimated to be A((14/15)Nx) < 0.07/0.1 MHz, A((13)Cx) < 0.1 MHz, corresponding to intrinsic couplings of about half these values. In parallel, we have explicitly calculated the hyperfine tensors for X = N-14/15/C-13/O-17, nuclear quadrupole coupling constant e(2)qQ for X = N-14, and hyperfine constants for the Fe sites of S = 3/2 FeMo-co using density functional theory (DFT) in conjuction with the broken-symmetry (BS) approach for spin coupling. If X = C/N, then the decoupling required by experiment strongly supports the "BS7' spin coupling of the FeMo-co iron sites, in which a small X hyperfine coupling is the result of a precise balance of spin density contributions from three spin-up and three spin-down (3 up arrow:3 down arrow) iron atoms of the [6Fe] prismane "waist" of FeMo-co; this would rule out the "BS6" assignment (4 up arrow:2 down arrow for [6Fe]) suggested in earlier calculations. However, even with the BS7 scheme, the hyperfine couplings that would be observed for X near g(2) are sufficiently large that they should have been detected: we suggest that the experimental results are compatible with X = N only if a(iso)(N-14/15(x)) < 0.03-0.07/0.05-0.1 MHz and ai(so)(C-13(X)) < 0.05-0.1 MHz, compared with calculated values of aiso(N-14/15(x)) = 0.3/0.4 MHz and a(iso)(C-13(X)) = 1 MHz. However, the DFT uncertainties are large enough that the very small hyperfine couplings required by experiment do not necessarily rule out X = N/C.