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) on nitrogenase and the mediators used during voltammetry experiments. Dithionite is routinely added to purification and storage buffers to protect nitrogenase and other anaerobic enzymes from oxygen. Dithionite has extremely complex chemistry with a myriad of decomposition products. Herein, 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. This cannot be distinguished from enzyme catalysis, particularly with cobaltocenium mediators which can result in amplified kobs values. 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 from the enzyme stock prior to performing cyclic voltammetry experiments. Dithionite, used for oxygen scavenging in protein storage buffers for anaerobic enzymes, is redox active in mediated enzyme electrochemistry of nitrogenase. ‘Pseudo’ catalysis is observed in the presence of dithionite which is hard to distinguish from nitrogenase catalysis. The effect is an amplified kobs value when performing foot of the wave analysis (FOWA).