Hydrogenases are widespread metalloenzymes used for the activation and production of molecular hydrogen. Understanding the catalytic mechanism of hydrogenases can help to establish industrial (bio)catalytic hydrogen production and conversion. Here we show the observation of so-far undetectable intermediates of [Fe]-hydrogenase in its catalytic cycle. We observed these intermediates by applying a signal-enhancing NMR technique based on parahydrogen. Molecular hydrogen occurs as orthohydrogen or parahydrogen, depending on its nuclear spin state. We found that catalytic conversion of parahydrogen by the [Fe]-hydrogenase leads to notably enhanced NMR signals (parahydrogen-induced polarization, PHIP). The observed signals encode information about how the [Fe]-hydrogenase binds hydrogen during catalysis. Our data support models of the catalytic mechanism that involve the formation of a hydride at the iron centre. Moreover, PHIP enabled studying the binding kinetics. This work demonstrates the hitherto unexploited power of PHIP to study catalytic mechanisms of hydrogenases.

氢化酶是广泛使用的金属酶，用于活化和产生分子氢。了解氢化酶的催化机制有助于建立工业（生物）催化氢气生产和转化。在这里，我们展示了在其催化循环中迄今为止无法检测到的 [Fe]-氢化酶中间体的观察结果。我们通过应用基于仲氢的信号增强 NMR 技术来观察这些中间体。氢分子以正氢或仲氢的形式存在，具体取决于其核自旋态。我们发现 [Fe]-氢化酶对对氢的催化转化导致 NMR 信号显著增强（对氢诱导极化，PHIP）。观察到的信号编码有关 [Fe]-氢化酶在催化过程中如何结合氢的信息。我们的数据支持涉及在铁中心形成氢化物的催化机制模型。此外，PHIP 能够研究结合动力学。这项工作证明了 PHIP 迄今为止未开发的能力，用于研究氢化酶的催化机制。