Establishing a Thermodynamic Landscape for the Active Site of Mo-Dependent Nitrogenase.

Nitrogenase enzymes are the only biological catalysts able to convert N to NH. Molybdenum-dependent nitrogenase consists of two proteins and three metallocofactors that sequentially shuttle eight electrons between three distinct metallocofactors during the turnover of one molecule of N. While the kinetics of isolated nitrogenase has been extensively studied, little is known about the thermodynamics of its cofactors under catalytically relevant conditions. Here, we employ a recently described pyrene-modified linear poly(ethylenimine) hydrogel to immobilize the catalytic protein of nitrogenase onto an electrode surface. The resulting electroenzymatic interface enabled direct measurement of reduction potentials associated with each metallocofactor of the nitrogenase complex, illuminating the role of nitrogenase reductase in altering the potential landscape in the active site of nitrogenase and revealing the endergonic nature of electron-transfer steps associated with the conversion of N to NH under physiological conditions.