Max Planck Institute for Terrestrial Microbiology, Marburg 35043, Germany.
PRESTO, Japan Science and Technology Agency (JST), Saitama 332-0012, Japan.
Nat Chem. 2015 Dec;7(12):995-1002. doi: 10.1038/nchem.2382. Epub 2015 Nov 2.
[Fe]-Hydrogenase catalyses the reversible hydrogenation of a methenyltetrahydromethanopterin substrate, which is an intermediate step during the methanogenesis from CO2 and H2. The active site contains an iron-guanylylpyridinol cofactor, in which Fe(2+) is coordinated by two CO ligands, as well as an acyl carbon atom and a pyridinyl nitrogen atom from a 3,4,5,6-substituted 2-pyridinol ligand. However, the mechanism of H2 activation by [Fe]-hydrogenase is unclear. Here we report the reconstitution of [Fe]-hydrogenase from an apoenzyme using two FeGP cofactor mimics to create semisynthetic enzymes. The small-molecule mimics reproduce the ligand environment of the active site, but are inactive towards H2 binding and activation on their own. We show that reconstituting the enzyme using a mimic that contains a 2-hydroxypyridine group restores activity, whereas an analogous enzyme with a 2-methoxypyridine complex was essentially inactive. These findings, together with density functional theory computations, support a mechanism in which the 2-hydroxy group is deprotonated before it serves as an internal base for heterolytic H2 cleavage.
[Fe]-氢化酶催化亚甲基四氢甲酰基喋呤底物的可逆加氢，这是 CO2 和 H2 甲烷化过程中的中间步骤。活性位点包含一个铁-鸟苷基吡啶醇辅因子，其中 Fe(2+)由两个 CO 配体、一个酰基碳原子和一个吡啶基氮原子配位，来自 3,4,5,6-取代的 2-吡啶醇配体。然而，[Fe]-氢化酶催化 H2 活化的机制尚不清楚。在这里，我们使用两种 FeGP 辅因子模拟物从脱辅基酶中重新组装 [Fe]-氢化酶，以创建半合成酶。小分子模拟物再现了活性位点的配体环境，但单独对 H2 结合和活化没有活性。我们表明，使用含有 2-羟基吡啶基团的模拟物重新组装酶可恢复活性，而具有类似 2-甲氧基吡啶络合物的类似酶则基本上没有活性。这些发现以及密度泛函理论计算支持一种机制，其中 2-羟基在作为异裂 H2 裂解的内部碱之前被去质子化。
