特征谱学方法研究固氮酶加氢催化活性中心

Nitrogenase catalyzes the reduction and hydrogenation of dinitrogen to ammonia in the process of biological nitrogen fixation. In the past few decades, its catalytic mechanism and chemical simulation have been widely studied. The high resolution X-ray structural analyses and the spectral studies of the MoFe protein in nitrogenase reveal the catalytic active site of iron molybdenum cofactor (FeMo-co) as a cage structure MoFe7S9C(R-homocit) with a central carbon atom (H4homocit = homocitric acid), where the homocitrate chelates with molybdenum by alpha-alkoxyl and alpha-carboxyl groups. In this project, based on the comparisons of our previous exact small molecular structures of vanadyl citrate, malate, and citramalate with the protein chelated structure of homocitrate, a protonated structure of the catalytic active site has been proposed as MoFe7S9C(R-Hhomocit) in nitrogenase. To give much strong and exact evidence for the protonated structure, different labeled and optical homocitrates will be synthesized. The homocitrate will be used for the preparations of optical and labeled molybdenum homocitrates and their homologs in different oxidation states, which are characterized with vibrational circular dichroism, optical Raman and nuclear resonant vibrational spectroscopies, structural analysis and theoretical calculation. The spectra of the model complexes will be compared with those of iron molybdenum cofactor of active site in nitrogenase. This will verify definitely the coordination environment of molybdenum atom in iron molybdenum cofactor as chelated structure of MoFe7S9C(R-Hhomocit) with alpha-hydroxyl and alpha-carboxyl groups, or MoFe7S9C(R-homocit) with alpha-alkoxyl and alpha-carboxyl groups. The chelated alpha-hydroxyl and carboxyl structure of catalytic active site of nitrogenase will served as the basis for the studies of catalytic mechanism of nitrogenase and play important roles in delivering proton and catalytic redox process of the cluster in N2 reduction and H2 hydrogenation. The verified hydrogen structure will be important for the study of catalytic mechanism in nitrogenase which remains unclear.

固氮酶催化氮分子还原加氢成氨，它的催化活性中心结构历经四十余年的结构生物学和谱学研究，似乎已经完全明确为MoFe7S9C(R-homocit)，其中高柠檬酸通过alpha-烷氧基和羧基与钼螯合。基于柠檬酸钒系列小分子螯合物的精确结构与高柠檬固氮酶配位模式的比较，我们曾提出酶催化活性中心MoFe7S9C(R-Hhomocit)的加氢结构。为了提供是否加氢的强有力实验依据，本项目拟合成不同同位素标记和光学活性的高柠檬酸，制备不同价态、同位素标记的手性高柠檬酸钼螯合物及其同系物，利用振动圆二色光谱、旋光拉曼光谱、核振能谱、结构分析和理论计算等手段，比较高柠檬酸钼模型螯合物和固氮酶铁钼辅基催化活性中心的谱学特征，证明固氮酶铁钼辅基催化活性中心alpha-羟基或者是alpha-烷氧基和羧基的螯合结构。加氢结构的确认将为固氮酶固氮加氢成氨的催化作用机理研究提供必要的基础，推动固氮酶研究深入开展。

固氮酶能在温和的条件下将空气中的氮气转化为可供生物利用的含氮化合物，地球表面每年通过生物固氮作用获得的氮约2亿吨，占全球氮资源的65%。固氮酶钼铁蛋白的结构和光谱研究表明，它的催化活性中心铁钼辅基(FeMo-co)的结构为MoFe7S9C(R-homocit)(Hhis)(cys) (H4homocit=高柠檬酸, Hhis=组氨酸, Hcys=半胱氨酸)。高柠檬酸通过α-烷氧基和α-羧基与钼原子双齿螯合，游离的β-和γ-羧基与周围的氨基酸残基和水分子通过氢键连接。研究表明，高柠檬酸对于稳定Mo原子和N2分子的还原是必需的，实验和理论分析表明它可能与质子或电子的传递、钼的转移和存储有关。当底物分子(N2或NNH2)络合在Fe位上与高柠檬酸形成氢键时，后者的羟基质子是理想的质子源。2014年，本课题组从具有加氢配位模式的羟基羧酸钒模拟物出发，对固氮酶高柠檬酸的配位模式进行了加氢修订。在本项目执行过程中，我们分别以蛋白质数据库新近的110个铁钼辅基和4个铁钒辅基为基础，利用我们得到的羟基羧酸钼、钒模型化合物的光谱、结构数据和理论计算结果，特别是固氮酶钼铁蛋白的N-甲基甲酰胺的提取产物铁钼辅基的红外(IR)和圆偏振振动光谱(VCD)，为固氮酶铁钼辅基中高柠檬酸质子化提供了间接和直接的实验依据。关键的数据有：羟基羧酸咪唑钼(IV)的α-烷氧基与钼的配位键长[Mo–O 1.999(7)av Å]与乳酸钼络合物α-羟基配位键长[2.204(4)av Å]对比显著缩短，后者与报道的36种钼铁蛋白的高柠檬酸钼结构数据 (Mo–Oav 2.272 Å)接近，由此为FeMo-co中高柠檬酸质子化的间接证据。直接证据有：配位的α-烷氧基C–O的红外伸缩振动在1080波数附近；质子化后，配位α-羟基C–OH的伸缩振动红移约30 ~ 50波数； 特别是VCD图谱，VCD振动频率范围确定为1100~1000波数。比较羟基羧酸钼如R-乳酸钼的ν(C–OH)和ν(C–O)特征振动峰，当乳酸α-羟基配位的去质子化后，ν(C–OH)将从高波数移动到低波数，即发生红移现象，在R-高柠檬酸钼和铁钼辅基也观察到相同的规律；结合密度泛函理论计算，在NMF提取的FeMo-co中，质子化发生在R-高柠檬酸的α-烷氧基上，这一结论进一步扩展到钼固氮酶的铁钼辅基上。