A wide range of micro-organisms express hydrogenases - highly active enzymes for oxidation of H2, or reduction of H+ to produce H2. The efficiency of H2/H+ interconversion by hydrogenases is providing inspiration for development of new catalysts for fuel cells or clean H2 production built from abundant metals, but this requires an intimate knowledge of how H2 is activated at hydrogenase active sites. The Vincent group are using a combination of electrochemical, spectroscopic and structural methods to understand hydrogenase active site chemistry.
Direct electrochemical methods, in which hydrogenases are adsorbed as a sub-monolayer film onto a graphite electrode, provide precise control over active site chemistry. In this approach called (protein film electrochemistry), adsorbed enzyme molecules exchange electrons directly with the electrode so that no electron-transfer mediators are required. The current at the electrode reports on the catalytic activity of the adsorbed hydrogenase at each applied potential. Protein film electrochemistry has provided much insight into the chemistry of hydrogenases.
We are using a range of coupled electrochemical and infrared spectroscopic approaches to reveal structural details of enzyme active site chemistry during catalytic turnover. The active sites of hydrogenases incorporate ligands that are unusual in biology: carbon monoxide (CO) and cyanide (CN-). These ligands provide a useful spectroscopic handle for studying structural changes at the metal centre because vibrational transitions associated with CO and CN- give rise to relatively intense absorption bands in the infrared.
More recently we have applied electrochemistry to crystals of hydrogenase. We have found that the redox state of the enzyme molecules in the crystal responds rapidly to a change at the electrode, allowing us to obtain crystals in each redox state. This has enabled us to obtain high resolution crystal structures of hydrogenase in each redox state relevant to catalysis. We are now combining this with advanced protein crystallography using x-ray free electron lasers and neutron diffraction.
We are now extending these studies to other metalloenzyme such as nitrogenase.
