The prototypical hydrogen-producing enzyme, the membrane-bound formate hydrogenlyase (FHL) complex from Escherichia coli, links formate oxidation at a molybdopterin-containing formate dehydrogenase to proton reduction at a [NiFe] hydrogenase. It is of intense interest due to its ability to efficiently produce H₂ during fermentation, its reversibility, allowing H₂-dependent CO₂ reduction, and its evolutionary link to respiratory complex I. FHL has been studied for over a century, but its atomic structure remains unknown. Here we report cryo-EM structures of FHL in its aerobically and anaerobically isolated forms at resolutions reaching 2.6 Å. This includes well-resolved density for conserved loops linking the soluble and membrane arms believed to be essential in coupling enzymatic turnover to ion translocation across the membrane in the complex I superfamily. We evaluate possible structural determinants of the bias toward hydrogen production over its oxidation and describe an unpredicted metal-binding site near the interface of FdhF and HycF subunits that may play a role in redox-dependent regulation of FdhF interaction with the complex.

原型产氢酶，即来自大肠杆菌的膜结合甲酸氢裂解酶 (FHL) 复合物，将含钼蝶呤的甲酸脱氢酶的甲酸氧化与 [NiFe] 氢化酶的质子还原联系起来。由于其在发酵过程中有效产生 H ₂的能力及其可逆性，允许 H ₂依赖 CO ₂还原及其与呼吸复合物 I 的进化联系。FHL 已被研究了一个多世纪，但其原子结构仍然未知。在这里，我们报告了分辨率达到 2.6 Å 的有氧和厌氧分离形式的 FHL 的冷冻电镜结构。这包括连接可溶臂和膜臂的保守环的良好分辨密度，这些环被认为对于将酶促转换与复合物 I 超家族中的跨膜离子易位偶联至关重要。我们评估了氢气生产偏向于其氧化的可能结构决定因素，并描述了 FdhF 和 HycF 亚基界面附近的一个不可预测的金属结合位点，它可能在 FdhF 与复合物相互作用的氧化还原依赖性调节中发挥作用。