An Efficient Viologen-Based Electron Donor to Nitrogenase

Nitrogenase catalyzes the reduction of N₂ to NH₃, supporting all biological nitrogen fixation. Electron donors to this enzyme are ferredoxin or flavodoxin (in vivo) and sodium dithionite (in vitro). Features of these electron donors put a limit on spectrophotometric studies and electrocatalytic applications of nitrogenase. Although it is common to use methyl viologen as an electron donor for many low-potential oxidoreductases, decreased nitrogenase activity is observed with an increasing concentration of methyl viologen, limiting its utility under many circumstances. In this work, we suggest that this concentration-dependent decrease in activity can be explained by the formation of a dimer of the radical cation of methyl viologen (Me₂V)₂ at higher methyl viologen concentrations. In addition, viologens functionalized with positively and negatively charged groups were synthesized and studied using spectroscopy and cyclic voltammetry. A sulfonated viologen derivative, 1,1'-bis(3-sulfonatopropyl)-4,4'-bipyridinium radical {[(SPr)₂V]^-}, was found to support full nitrogenase activity up to a mediator concentration of 3 mM, while the positively charged viologen derivative was not an efficient reductant of nitrogenase due to the high standard redox potential. The utility of [(SPr)₂V]^- as an electron donor for nitrogenase was demonstrated by a simple, sensitive spectrophotometric assay for nitrogenase activity that can provide accurate values for the specific activity and turnover rate constant under argon. Under N₂, the formation of ammonia was confirmed. Because of the observed full activity of nitrogenase and low overpotential, [(SPr)₂V]^- should also prove to be valuable for nitrogenase electrocatalysis, including bioelectrosynthetic N₂ reduction.