Development of an infrared spectroscopic approach for studying metalloenzyme active site chemistry under direct electrochemical control

Direct electrochemical methods have been productive in revealing mechanistic details of catalysis by a range of metalloenzymes including hydrogenases and carbon and nitrogen cycling enzymes. In this approach, termed protein film electrochemistry, the protein is attached or adsorbed on the electrode surface and exchanges electrons directly, providing precise control over redox states or catalysis and avoiding diffusion-limited electron transfer. The 'edge' surface of pyrolytic graphite has proved to be a particularly good surface for adsorption of proteins in electroactive conformations. We now describe development of an approach that combines the precise control achieved in direct electrochemical measurements at a graphite electrode with surface infrared (IR) spectroscopic analysis of chemistry occurring at metallocentres in proteins. Hydrogenases are of particular interest: their unusual organo-metallic active sites - iron or nickel-iron centres coordinated by CO and CN- - give rise to IR v(CO) and v(CN) bands that are detected readily because these ligands are strong vibrational oscillators and are sensitive to changes in electron density and coordination at the metals. Small diatomic species also bind as exogenous ligands (as substrate, product, activator or inhibitor) to a range of other important metalloproteins, and understanding their reactivity and binding selectivity is critical in building up a multidimensional picture of enzyme chemistry and evolutionary history. The surface IR spectroelectrochemical approach we describe is based around Attenuated Total Reflectance (ATR) mode sampling of a film of pyrolytic graphite particles modified with a protein of interest. The particle network extends the electrode into three-dimensional space, providing sufficient adsorbed protein for spectroscopic analysis under precise electrochemical control. This strategy should open up new opportunities for detection of redox-dependent chemistry at metal centres in proteins, including short-lived catalytic intermediates and time-resolved details of catalysis and inhibition.