Anoxic Enzymatic Conversion of Acetylene

气体乙炔（乙炔）在自然界中经过两种已知的酶转化。氮酶是将二氮还原固定为生物可利用铵所需的酶，它通过双电子步骤将乙炔还原为乙烯（乙烯）。相反，钨/ [4Fe:4S]酶乙炔水合酶催化气体水合生成乙醛。这两种蛋白质在结构和功能上都得到了很好的表征，但关于乙炔结合的模式和确切位点及其转化机制仍然存在很大的争议。显然，乙炔转化的机制遵循完全不同的途径：虽然氮酶最有可能将乙炔（和其他底物）结合到[Mo:7Fe:9S:X]：高柠檬酸FeMo辅因子上，低氧化还原电位的电子从该辅因子直接转移到底物上，但钨蛋白乙炔水合酶有一个独特的结合袋，由疏水氨基酸环预先形成。这里的反应可能不涉及金属-碳键的形成，而是与钨配合的水分子的活化，随后攻击炔的三键。我们将在实验室中建立和优化天然和重组乙炔水合酶的生产，创建点特异性突变体，用于酶活性分析，并获得野生型和变异蛋白的高分辨率结构，这些蛋白被乙炔或其他候选配体加压。本文将从紫氏固氮菌中分离出固氮酶，并测定其还原乙炔的活性。乙炔，特别是强配体CO将用于加压实验，以获得原子分辨率数据，我们期望从中获得重要的机制线索。

The gas acetylene (ethyne) undergoes two known enzymatic conversions in nature. Nitrogenase, the enzyme required for the reductive fixation of dinitrogen into bioavailable ammonium, reduces acetylene in a two-electron step to ethylene (ethene). In contrast, the tungsten / [4Fe:4S] enzyme acetylene hydratase catalyzes the hydratation of the gas to yield acetaldehyde. The proteins are well characterized structurally and functionally, but the mode and exact site of acetylene binding and the mechanism of its conversion remain under heavy debate for both cases. Clearly, the mechanisms of acetylene conversion follow entirely different routes: while nitrogenase most likely binds acetylene (and other substrates) to the [Mo:7Fe:9S:X]:homocitrate FeMo cofactor, from which electrons of a low redox potential get transferred directly to the substrate, the tungstoprotein acetylene hydratase has a distinct, binding pocket pre-formed by a ring of hydrophobic amino acids. Here the reaction may not involve the formation of a metal-carbon bond, but rather the activation of a water molecule coordinated to tungsten that subsequently attacks the triple bond of the alkyne. We will establish and optimize both native and recombinant production of acetylene hydratase in our laboratory, create point-specific mutants that will be analyzed for enzymatic activity and obtain high-resolution structures of wild type and variant proteins pressurized with acetylene or other candidate ligands. Nitrogenase will be isolated from Azotobacter vinelandii and assayed for acetylene reduction activity. Acetylene, and in particular the strong ligand CO will be used in pressurization experiments to obtain atomic resolution data from which we expect to gain essential mechanistic clues.