Department of Chemistry, Northwestern University, Evanston, Illinois 60208, USA.
J Am Chem Soc. 2010 Mar 3;132(8):2526-7. doi: 10.1021/ja910613m.
We here report the first direct evidence addressing the possible involvement of Mo in substrate interactions during catalytic turnover. When the alpha-70(Ile) MoFe protein is freeze-trapped during H(+) reduction under Ar, the majority of the resting state EPR signal from the molybdenum-iron cofactor (FeMo-co) disappears and is replaced by the S = 1/2 signal of an intermediate that has been shown to be the E(4) MoFe state, which is activated for N(2) binding and reduction through the accumulation of 4 electrons/protons by FeMo-co. ENDOR studies of E(4) showed that it contains two hydrides bound to FeMo-co. We calculate that Mo involvement in hydride binding would require a vector-coupling coefficient for Mo of |K(Mo)| approximately > 0.2 and determine K(Mo) for the E(4) intermediate state through 35 GHz ENDOR measurements of a (95)Mo enriched MoFe protein, further comparing the results with those for the E(0) resting state. The experiments show that Mo of the resting-state FeMo-co is perturbed by the alpha-70(Ile) substitution and that the isotropic (95)Mo hyperfine coupling in E(4) is a(iso) approximately 4 MHz, less than that for the resting state. The decrease in a(iso) for (95)Mo of E(4) from the already small value in the resting state MoFe protein strongly suggests that the resting Mo(IV) is not one-electron reduced during the accumulation of the four electrons of E(4). In any case, the effective K for Mo is very small; |K(Mo)| approximately < 0.04, at least 5-fold less than the lower bound required for Mo to be involved in forming a Mo-H-Fe, hydride. As the hydride couplings also are both far too small and of the wrong symmetry to be associated with a terminal hydride on Mo, we may thus conclude that Mo does not participate in binding a hydride of the catalytically central E(4) intermediate and that only Fe ions are involved. Nonetheless, the response of the Mo coupling to subtle conformational changes in E(0) and to the formation of E(4) suggests that Mo is intimately involved in tuning the geometric and electronic properties of FeMo-co in these states.
我们在此报告了首个直接证据，证明钼可能参与了催化循环过程中底物的相互作用。当在氩气下进行 H+还原时，α-70（异亮氨酸）MoFe 蛋白被冷冻捕获，FeMo-co 的大部分静止状态 EPR 信号消失，取而代之的是已被证明为 E(4)MoFe 状态的中间体的 S = 1/2 信号，该状态通过 FeMo-co 积累 4 个电子/质子而被激活，从而可以与 N2 结合并还原。E(4)的 ENDOR 研究表明，它含有两个与 FeMo-co 结合的氢化物。我们计算出，Mo 参与氢化物结合需要 Mo 的矢量耦合系数|K(Mo)|大于 0.2，并通过对富含(95)Mo 的 MoFe 蛋白进行 35GHz ENDOR 测量，确定了 E(4)中间态的 K(Mo)，进一步将结果与 E(0)静止态的结果进行比较。实验表明，α-70（异亮氨酸）取代使 FeMo-co 的 Mo 受到干扰，并且 E(4)中的各向同性(95)Mo 超精细耦合 a(iso)约为 4MHz，小于静止态的 a(iso)。E(4)中(95)Mo 的 a(iso)相对于静止态 MoFe 蛋白中已经很小的 Mo 的减小强烈表明，在 E(4)中积累的四个电子期间，静止的 Mo(IV)没有被单电子还原。在任何情况下，Mo 的有效 K 都非常小；|K(Mo)|约<0.04，至少比 Mo 参与形成 Mo-H-Fe 氢化物所需的下限小 5 倍。由于氢化物偶合也太小且对称性错误，不能与 Mo 上的末端氢化物相关，因此我们可以得出结论，Mo 不参与催化中心 E(4)中间体的氢化物结合，只有 Fe 离子参与。尽管如此，Mo 偶合对 E(0)中微妙的构象变化和 E(4)形成的响应表明，Mo 密切参与了这些状态下 FeMo-co 的几何和电子性质的调谐。
