Quantitative Estimation of Nitrogenase Activity: Acetylene Reduction Assay

Abstract

Symbiotic nitrogen fixation plays an important role in agriculture, and there has been a goal to improve symbiotic efficiency to reduce the use of chemical fertilizers. Symbiotic nitrogen fixation is catalyzed by the molybdenum nitrogenase enzyme. The molybdenum nitrogenase is composed of MoFe protein (NifDK) and Fe protein (NifH). The MoFe protein is an α₂β₂ heterotetramer that contains the iron–molybdenum cofactors (FeMo-co) and P clusters. The FeMo-co is a [Mo–7Fe–9S–C-homocitrate] cluster which serves as the active site of nitrogen binding and reduction. The P-cluster is a [8Fe–7S] cluster which shuttles electrons to the FeMo-co. The Fe protein is a γ₂ homodimer bridged by an intersubunit [4Fe–4S] cluster that serves as the obligate electron donor to the MoFe protein. Nitrogenase is a versatile enzyme capable of catalyzing the reduction of several substrates other than nitrogen, including acetylene, azide, nitrous oxide, nitriles, and isonitriles. Ethylene produced due to the reduction of acetylene by nitrogenase is determined by using gas chromatography.