【作者】 赵德华；

【导师】 李季伦；

【作者基本信息】 中国农业大学 ， 微生物学， 2004， 博士

【摘要】 尽管当前人们对固氮酶的结构和功能的认识已达到了原子水平，但对固氮酶活性中心FeMoco中催化底物N₂和H⁺的还原位点，及用于还原反应的质子和电子通道，至今仍未确定。 本论文中，通过定点突变及基因替代技术，成功构建了四个K. pneumoniae固氮酶突变体MoFe蛋白，其中：两个为单突变体，将野生型固氮酶钼铁蛋白α亚基中，铁钼辅因子（FeMoco）周围多肽链的Gln α 190和His α 194分别置换为Lys和Gln(α-Lys¹⁹⁰ Kp1和α-Gln¹⁹⁴ Kp1)；两个为双重置换的突变体，将上述两个氨基酸单独替换突变分别引入nifV突变株（nifV^-，与野生型FeMoco共价连接的高柠檬酸被置换为柠檬酸）而获得(α-Lys¹⁹⁰／NifV^- Kp1和α-Gln¹⁹⁴／NifV^- Ko1)。 在固氮条件下，所构建固氮酶突变菌株均表现为严格的Nif^-表型，不能进行固氮生长。大量培养各突变株并充分诱导细胞固氮酶的表达，利用相同的柱层析和制备电泳程序纯化了四个突变体固氮酶以及来自野生型和nifV突变株的MoFe蛋白及野生型Fe蛋白。对比分析了四个突变体固氮酶、野生型和NifV^-固氮酶的MoFe蛋白催化N₂、H⁺及C₂H₂还原的酶学特性，及部分突变体固氮酶MoFe蛋白放H₂和C₂H₂还原受CO抑制的酶动力学，并推测出N₂和H⁺还原的位点。 四个突变体固氮酶、野生型和NifV^-固氮酶的MoFe蛋白的底物还原特性比较表明：（1）α-Gln¹⁹⁴替换不影响固氮酶还原质子时的总电子流；尤其引人注意的是，含有α-Lys¹⁹⁰和柠檬酸双重替换的MoFe蛋白几乎完全失去了质子还原的能力。（2）α-Gln¹⁹⁴替换与其它两个替换相比，对固氮酶N₂还原的影响更为直接。（3）α-Gln¹⁹⁴替换使得固氮酶还原C₂H₂时，电子流分配偏向于放氢，而α-Lys¹⁹⁰替换则完全阻断了固氮酶还原C₂H₂的电子流。CO对NifV^-MoFe蛋白放氢的最大抑制率为71％，α-Gln¹⁹⁴和柠檬酸双重替换使的CO抑制固氮酶放氢的最大抑制率下降到31％。（5）CO与含有α-Lys¹⁹⁰替换的MoFe蛋白的亲和力比对含有α-Gln¹⁹⁴和citrate双重替代的MoFe蛋白及NifV^-蛋白的亲和力要低。 对四个突变株细胞的C₂D₂还原特性及还原产物中反式-／顺式-1，2-二氘代乙炔的比例进行了测定并与野生型及nifV突变株相比较，结果表明只有α-Gln¹⁹⁴替换不影响C₂D₂还原产物中反式-／顺式-1，2-二氘代乙炔的比例，即未改变固氮酶还原C₂H₂加氢的立体构型的专一性。 各突变体固氮酶底物还原及酶动力学特性的变化暗示：（1）α-Lys¹⁹⁰位点及其与高柠檬酸的协同作用，在质子和电子向FeMoco传递中起重要作用，而α-Gln¹⁹⁴和高柠檬酸位点的改变对固氮酶放氢的影响却相对独立。（2）α-Gln¹⁹⁴位点在N₂还原中有其特有的功能，并体现出在H⁺和N₂，或H⁺和C₂H₂还原时调节电子分配的角色。（3）α-Lys¹⁹⁰位点和高柠檬酸位点可能通过一种共有的机制影响固氮酶FeMoco上一个放氢位点。 根据本论文的研究结果，我们推测固氮酶络合和还原N₂和H⁺的位点：（1）固氮酶FeMoco中央的Fe原子区为N₂络合和还原的位点，所消耗的质子和电子由临近的α-His¹⁹⁴通过与FeMoco的一个S原子（S2B）之间形成的氢键进入，其反应式为：N₂+8H⁺+8e^-→2NH₃+HZ，此反应受CO抑制;（2）几Moco的Mb原子为专一的放氢位点，所用的质子和电子由与M。结合的高柠橡酸传入，其反应式为:ZH++2e’，HZ，此反应不受co抑制;。心Inl90一高柠稼酸一M。是一条通向FeMoco的重要的电子传递途径。 同时进一步验证了本实验室以前提出的固氮酶在生理条件下具有双位点放氢的模式，并明确固氮酶双位点放氢模式为:放氢位点I:为依赖NZ的放氢位点，定位于FeMOCo的中间六铁区，同时是NZ、CZHZ和CO的络合位点:放氢位点H:为不依赖NZ的放氢位点，定位于FeMoco的Mo原子上。放氢位点11即M。位是FeMoc。中接受电子和质子的首要位点。 本论文中所构建的含价Lysl90和citrate，以及。心Inl侧和ci姗双重替换的突变体固氮酶在进一步固氮酶机制的研究中将会非常有用。更多还原

【Abstract】 The researches of nitrogenase have reached the atomic levels, but where the substrates bind and are reduced at the FeMoco remains unknown and the pathway of electron and proton transfer to FeMoco in the enzyme catalysis is also undetermined.In this research, site-directed mutagenesis and gene replacement procedures were used to construct two doubly altered MoFe proteins with a-Lysine 190 (a-Lys190) and a-Glutamine 194 (a-Gln194) substitutions and with a second substitution of citrate for homocitrate of FeMoco (from a nijV mutant) (a-Lys190/NifV- Kpl a-Gln194/NifV- Kp1) and two singly altered MoFe proteins with only a-Lysine 190 and a-Glutamine 194 substitutions (a-Lys190 Kpl and a-Gln194 Kpl) from Klebsiellapneumoniae. All four mutants expressing altered nitrogenases exhibited strictly Nif phenotype under N2-fixation condition and failed to grow diazotrophically. An auto-controlled fermentor was used to culture these strains under nitrogen fixation conditions and the nitrogneases were effectively depressed. Four altered MoFe proteins, together with WT and NifV" MoFe proteins and the WT Fe protein were purified by an identical procedure using chromatography and preparative electrophoresis. The activities of N2, H+ and C2H2 reduction catalyzed by these four MoFe proteins were detected and kinetics of H2 evolution and C2H2 inhibited by CO for some special altered MoFe proteins were also analyzed, which makes us possible inquire the N2 and H+ binding and reduced sites.Comparison of substrate-reduction properties of these altered MoFe proteins with WT and NifV-MoFe proteins showed that: (1) only the a-Gln194 substitution did not affect the total electron to proton reduction and notably double altered MoFe protein with substitution of citrate and a-Lys190 only retained very poor proton reduction activity. (2) a-Gln194 substitution made a more direct effect on N2 reduction then other two substitutions. (3) Substitution of a-Gln194 deflected electron to H2 evolution and substitution of a-Lys190 almost blocked the electron transfer to C2H2 reduction. (4) H2 evolution by MoFe protein with a-Gln194 and citrate substitutions was inhibited by CO at 32% inhibition rate, comparing to 71% of that for NifV- MoFe protein. (5) CO has a more weak affinity to MoFe protein with a-Lys190 substitutions then to NifV- MoFe protein or MoFe protein with a-Gln194 and citrate substitutions.Whole cell C2D2 reduction by all four mutants comparing to wild type and ni/V mutant was also detected. The result showed that only single a-Gln194 substitution did not perturb the stereospecificity of protonation of C2D2.The above comparing results indicate that in MoFe protein (1) a a-Gln190 site and its association with homocitrate are important for the transfer of electron/proton to FeMoco, while a-His194 site and the homocitrate are independent in H2 evolution. (2) a-Gln194 has a special role in the N2 reduction and acts as a regulatory site of electron distribution between H2 and NH3or between H2 and C2H4; (3) a-Glu190 and homocitrate share some common mechanism to affect a H2 evolution site of FeMoco.According the implications obtained in this study, the N2 and H+ binding and reduced model atFeMoco was proposed : (1) N2 binds and is reduced at the central Fe region of FeMoco, the electron and proton for N2 reduction is transferred from S2B atom of FeMoco via ct-Hism, the reaction is N2 + 8H+ +8e→ 2NH3 + H2, and is inhibited by CO. (2) Mo of FeMoco is occupied by an obligatory H2 evolution and the electron and proton to Mo is transferred from homocitrate, the reaction is 2H+ + 2e→ H2, and is escaped the inhibition by CO. a-Gln190 -omocitrate-Mo is an important electron transfer pathway to FeMoco.This N2 and H+ binding and reduced model also developed our early proposed two-site H2 evolution model for nitrogenase: Site I, N2-dependent H2 evolution site, is provided by the central six-Fe region of FeMoco, which is also provided the N2, C2H2, CO binding site. Site II, the N2-independent H2 evolution site, is located at the Mo更多还原