| Denitrification is a key pathway in the global nitrogen cycle. It comprises a series of reductions from nitrate through nitrite, nitric oxide, nitrous oxide and finally to dinitrogen gas. This dissimilatory pathway is carried out by soil, marine, and freshwater organisms, removing nitrogen from the biosphere and returning it to the atmosphere. Understanding the steps of the pathway on a molecular level could have a substantial impact on agriculture, control of air pollution, as well as on removal of nitrate pollution from water.; The focus of this research was on the final reduction of nitrous oxide (N2O) to dinitrogen gas by the enzyme nitrous oxide reductase (N 2OR). This work involved purification and characterization of N 2OR as well as some of its accessory proteins, which are required for production of active enzyme containing all of the necessary copper. N 2OR was purified in several different forms, each containing different amounts of copper, different levels of activity, and showing different spectroscopic characteristics. N2OR is a homodimer containing two Cu sites per monomer. These are the binuclear, CuA electron transfer site and the novel, tetranuclear, sulfide-bridged CuZ catalytic site.; The genes coding for the accessory proteins are organized into three separate transcriptional units in a cluster with the nosZ (N2OR) gene. These units are nosR, nosDFYL, and nosX. The accessory protein NosX, which is speculated to be involved in either assisting regulation of N 2OR synthesis or loading of the CuA site in N2OR, was purified and shown to be a flavoprotein containing FAD as the cofactor. Another auxilliary protein, NosD, was purified with two other proteins (one being N2OR) tightly bound to it. This work begins the elucidation of the roles of these accessory proteins in N2OR processing. |
