Temperature-Dependent N2 Reduction and Energy Demands of Mo- and V-Nitrogenases

Abstract

Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen to ammonia, a key step in the global nitrogen cycle. The Mo- and V-dependent nitrogenase isozymes are expressed under different environmental conditions, including temperature, yet little is known about how their catalytic behavior changes with temperature. Here, we examine the temperature dependence of total electron flux, electron partitioning into the products NH3 and H2, and estimate ATP energy costs per N2 fixed for purified Mo- and V-nitrogenases from Azotobacter vinelandii over a 10-50 ˚C temperature range. Both isozymes exhibit similar temperature-dependent scaling of total electron flux to all products across the temperature range, but with significantly different distributions of electrons to H2 or NH3 formation. As temperature increases, V-nitrogenase diverts an increasing fraction of electron flux into H2 formation, whereas Mo-nitrogenase maintains a nearly constant electron partitioning to N2 reduction. The estimated ATP molecules hydrolyzed per N2 reduced change little for the Mo-nitrogenase across this temperature range, but rises significantly for the V-nitrogenase with increasing temperature, reaching over 200 ATP per N2 fixed at 50 ˚C. These results demonstrate that, in A. vinelandii, the Mo- and V-nitrogenases exhibit significantly different responses of electron partitioning to products and ATP hydrolyzed per N2 reduced to changes in temperature. These findings suggest environmental conditions under which each isozyme would be optimal, with V- and Mo-nitrogenase both being favorable at low temperatures and Mo-nitrogenase being the only energetically reasonable enzyme at higher temperatures.