Enhancing and Stabilizing Hydrogen Catalysis Through [NiFe]‐Hydrogenase Immobilization Within Macroporous Covalent Organic Frameworks

Abstract [NiFe]‐hydrogenases are highly efficient metalloenzymes catalyzing the reversible conversion of H ₂ into protons and electrons. These enzymes hold immense potential for sustainable biohydrogen production, as well as their integration into biotechnological devices. However, their inherent instability and the challenges associated with heterogeneous integration necessitate robust immobilization strategies. Covalent organic frameworks (COFs), with their high surface areas, tunable pore functionality, and chemical stability, offer a compelling platform for enzyme immobilization, potentially enhancing enzyme loading, stability, and catalytic performance. To address the limitations related to enzyme immobilization strategies, the membrane‐bound hydrogenase (MBH) from Cupriavidus necator is encapsulated within two different water‐stable β ‐ketoenamine COFs functionalized with sulfonic acid (─SO ₃ H) and carboxylic acid (─COOH) groups, respectively. These functional groups are strategically chosen to influence enzyme orientation and to allow electronic connection between enzyme and electrode. The COFs, featuring hierarchical pore sizes with macropores (≈80 nm) generated via a hard‐templating method, provide optimized enzyme stabilization, enhance proton and electron transport, and facilitate interactions promoting efficient charge transfer. Notably, MBH confined within these COFs exhibits not only enhanced electron transfer activity toward an electrode but also improved stability.