Aerobic Photocatalytic H2 Production by a [NiFe] Hydrogenase Engineered to Place a Silver Nanocluster in the Electron Relay

Hydrogenase-1 (Hyd-1) from E. coli poses a conundrum regarding the properties of electrocatalytic reversibility and associated bidirectionality now established for many redox enzymes. Its excellent H-2-oxidizing activity begins only once a substantial overpotential is applied, and it cannot produce H-2. A major reason for its unidirectional behavior is that the reduction potentials of its electron- relaying FeS clusters are too positive relative to the 2H(+)/H-2 couple at neutral pH; consequently, electrons held within the enzyme lack the energy to drive H-2 production. However, Hyd-1 is O-2-tolerant and even functions in air. Changing a tyrosine (Y) or threonine (T), located on the protein surface within 10 angstrom of the distal [4Fe-4S] and medial [3Fe-4S] clusters, to cysteine (C), allows site-selective attachment of a silver nanocluster (AgNC), the reduced or photoexcited state of which is a powerful reductant. The AgNC provides a new additional redox site, capturing externally supplied electrons with sufficiently high energy to drive H-2 production. Assemblies of Y'227C (or T'225C) with AgNCs/PMAA (PMAA = polymethyl acrylate templating several AgNC) are also electroactive for H-2 production at a TiO2 electrode. A colloidal system for visible-light photo-H-2 generation is made by building the hybrid enzyme into a heterostructure with TiO2 and graphitic carbon nitride (g-C3N4), the resulting scaffold promoting uptake of electrons excited at the AgNC. Each hydrogenase produces 40 molecules of H-2 per second and sustains 20% activity in air.