The effect of dithionite and its decomposition products on redox mediators used in the cyclic voltammetry of nitrogenase enzymes

Cyclic voltammetry is a powerful tool to study enzyme mechanisms. Over the last decade voltammetry has been applied to probe aspects of nitrogenase catalysis. One aspect that is often overlooked is the effect of dithionite (S2O42-, DTH) that is routinely added to purification and storage buffers to protect nitrogenase and anaerobic enzymes alike (e.g hydrogenase) from oxygen. Dithionite has extremely complex chemistry with a myriad of decomposition products. Here we sought to systematically investigate the effect of dithionite and some of its decomposition products on the voltammetry of different redox mediators independently and in conjunction with nitrogenase. We found the major decomposition product sulfite (SO32-) gives rise to reductive catalysis, which cannot be distinguished from enzyme catalysis, particularly with cobaltocenium mediators. We provide recommendations on how to identify and avoid interpreting ‘pseudo’ catalysis in lieu of enzyme catalysis by DTH and reinforce the requirement to remove DTH prior to performing cyclic voltammetry experiments.