A Voltammetric Study of Nitrogenase Catalysis Using Electron Transfer Mediators

Nitrogenase catalyzes the reduction of an array of small molecules, including N 2 to NH 3, by delivering electrons and protons to substrates bound to the active site metal cluster FeMo-cofactor. A challenge in describing the mechanism of nitrogenase-catalyzed reduction reactions is quantifying electron flow through the enzyme to different substrates. In this study, a mediated cyclic voltammetry approach was developed that provides a quantitative analysis of electron flow through nitrogenase. Conditions were optimized to reveal the catalytic reaction rate-limiting step. Analysis of the current response by an electrochemical approach yielded a catalytic rate constant ( k cat ) of 14 s –1, consistent with earlier studies. The current approach was used to resolve a long-standing conundrum in nitrogenase research, the apparent inhibition of electron flow through nitrogenase with increasing partial pressures of N 2 . It was demonstrated using this voltammetric approach in the absence of the reductant dithionite that total electron flow through nitrogenase remains constant up to a N 2 partial pressure of 1 atm.