Nitrogenases are remarkably able to break the triple bond of N2 to produce NH3 under ambient temperature and pressure in contrast to the extreme conditions required by the Haber-Bosch process. Biological ammonia production is very energy intensive, consuming at least 16 molecules of ATP for every N2 reduced (~ 13 GJ ton−1 of ammonia, assuming 28 kJ mol−1 produced by hydrolysis of ATP to ADP).
The binding of ATP to nitrogenase is known to induce conformational changes, promoting electron transfer and altering the redox potentials of key metal clusters. Release of phosphate, the product ATP hydrolysis, has been established as the rate limiting step of catalysis. There are however still many unanswered questions regarding the exact role of ATP in nitrogenase catalysis, especially why nitrogenase requires so much ATP.
Cyclic voltammetry is a powerful technique to probe enzymatic mechanisms, especially when the shape of the voltammogram is simulated. The natural electron donor of nitrogenase can be readily replaced by redox mediators, such as viologens and cobaltocenes, which shuttle electrons from the electrode to an enzyme.
We have carried out a detailed voltametric study of nitrogenase catalysis and simulated the experimentally obtained voltammograms to probe the role of ATP in nitrogenase. Our results shed light on the rates of ATP binding, a step which has been largely unexplored to date.