A conformational equilibrium in the nitrogenase MoFe protein with an α-V70I amino acid substitution illuminates the mechanism of H 2 formation
A conformational equilibrium in the nitrogenase MoFe protein with an α-V70I amino acid substitution illuminates the mechanism of H 2 formation
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固氮酶 MoFe 蛋白中α-V70I 氨基酸取代的构象平衡阐明了 H 2 形成的机制
DOI：
10.1039/d2fd00153e
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发表时间：
2023
影响因子：
3.4
通讯作者：
Hoffman, Brian M.
中科院分区：
文献类型：
--
作者：
Lukoyanov, Dmitriy A.;Yang, Zhi-Yong;Shisler, Krista;Peters, John W.;Raugei, Simone;Dean, Dennis R.;Seefeldt, Lance C.;Hoffman, Brian M.
对α-V70 I-取代固氮酶MoFe蛋白的研究表明，FeMo-辅因子的Fe 6（Fe 7S 9 MoC-homocitrate）是N2结合/还原的关键位点。在Ar转换过程中冷冻捕获这种酶，捕获了高占有率的关键催化中间体，表示为E4（4 H），它积累了4[e−/H+]作为两个桥连基团，Fe 2-H-Fe 6和Fe 3-H-Fe 7，以及与两个硫结合的质子。E4（4 H）准备结合/还原N2，这是由机械耦合的H2还原消除N2驱动的。这个过程必须与正在进行的氢化物质子化（HP）竞争，当酶弛豫到E2（2 H）状态时释放H2，其中含有2[e−/H+]作为氢化物和硫结合的质子; E4（4 H）在α-V70 I中的积累被HP抑制增强。EPR和95 Mo ENDOR谱表明，α-V70 I酶在溶液和结晶状态下存在两种构象，一种是野生型（WT）样FeMo-co，另一种是扰动FeMo-co。这反映了Ile残基的两种构象，这在α-V70 I的X射线衍射数据的再分析中得到了可视化，并通过计算得到了证实。EPR测量显示2[e−/H+]传递到WT MoFe蛋白的E0状态和两种α-V70 I构象，产生含有Fe 3-H-Fe 7桥接氢化物的E2（2 H）;另一个2[e−/H+]的积累产生E4（4 H），Fe 2-H-Fe 6作为第二个氢化物。WT酶中的E4（4 H）和QM/MM计算显示的少数α-V70 I E4（4 H）构象通过两个HP步骤松弛至静息状态，这两个HP步骤逆转了形成过程：Fe 2-H-Fe 6的HP，然后是Fe 3-H-Fe 7的较慢HP，这导致含有Fe 3-H-Fe 7的E2（2 H）的瞬时积累。在主要的α-V70 I E4（4 H）构象中，Fe 2-H-Fe 6的HP被Ile侧链的定位被动抑制; Fe 3-H-Fe 7的缓慢HP首先发生，并且所得的E2（2 H）含有Fe 2-H-Fe 6。正是E4（4 H）中的这种HP抑制使得α-V70 I MoFe能够以高占有率积累E4（4 H）。此外，α-V70 I E4（4 H）中的HP抑制在动力学上揭示了无N2结合的氢化物还原消除，这是WT酶中排除的过程。
Study of α-V70I-substituted nitrogenase MoFe protein identified Fe6 of FeMo-cofactor (Fe7S9MoC-homocitrate) as a critical N2 binding/reduction site. Freeze-trapping this enzyme during Ar turnover captured the key catalytic intermediate in high occupancy, denoted E4(4H), which has accumulated 4[e−/H+] as two bridging hydrides, Fe2–H–Fe6 and Fe3–H–Fe7, and protons bound to two sulfurs. E4(4H) is poised to bind/reduce N2 as driven by mechanistically-coupled H2 reductive-elimination of the hydrides. This process must compete with ongoing hydride protonation (HP), which releases H2 as the enzyme relaxes to state E2(2H), containing 2[e−/H+] as a hydride and sulfur-bound proton; accumulation of E4(4H) in α-V70I is enhanced by HP suppression. EPR and 95Mo ENDOR spectroscopies now show that resting-state α-V70I enzyme exists in two conformational states, both in solution and as crystallized, one with wild type (WT)-like FeMo-co and one with perturbed FeMo-co. These reflect two conformations of the Ile residue, as visualized in a reanalysis of the X-ray diffraction data of α-V70I and confirmed by computations. EPR measurements show delivery of 2[e−/H+] to the E0 state of the WT MoFe protein and to both α-V70I conformations generating E2(2H) that contains the Fe3–H–Fe7 bridging hydride; accumulation of another 2[e−/H+] generates E4(4H) with Fe2–H–Fe6 as the second hydride. E4(4H) in WT enzyme and a minority α-V70I E4(4H) conformation as visualized by QM/MM computations relax to resting-state through two HP steps that reverse the formation process: HP of Fe2–H–Fe6 followed by slower HP of Fe3–H–Fe7, which leads to transient accumulation of E2(2H) containing Fe3–H–Fe7. In the dominant α-V70I E4(4H) conformation, HP of Fe2–H–Fe6 is passively suppressed by the positioning of the Ile sidechain; slow HP of Fe3–H–Fe7 occurs first and the resulting E2(2H) contains Fe2–H–Fe6. It is this HP suppression in E4(4H) that enables α-V70I MoFe to accumulate E4(4H) in high occupancy. In addition, HP suppression in α-V70I E4(4H) kinetically unmasks hydride reductive-elimination without N2-binding, a process that is precluded in WT enzyme.