Azurins With Tunable Reduction Potentials as Redox Mediators for Extracellular Electron Transfer

Electroactive microbes, such as Shewanella oneidensis MR-1, have been harnessed for a variety of bioelectrochemical and catalytic applications. Efforts to improve the electrochemical performance of these bacteria have included the use or biological synthesis of small-molecule redox mediators, such as flavins. However, limited redox range and complex biosynthetic pathways have restricted their broad applications. To overcome these limitations, we report the use of azurin, a cupredoxin commonly used as electron transfer proteins, as a redox mediator for S. oneidensis extracellular electron transfer (EET). We showed that S. oneidensis reduces four different azurin variants, spanning a 440 mV range, and that the reduction rate is dependent on azurin concentration (K_M = 28 ± 9 µM) and redox potential. We then applied these findings to several different bio- and electrosynthetic applications. We utilized azurin as a mediator in multiple EET-driven biocatalytic reactions, including atom transfer radical polymerization and azo dye decolorization. Finally, we showed that the inclusion of exogenous azurin improves current generation in microbial fuel cells. These results demonstrate the potential advantages of engineered redox proteins possess over small molecules for use in EET-driven applications.