Redox-controlled dinitrosyl formation at the diiron-oxo center of NorA.

In the denitrifying bacterium Ralstonia eutropha H16, the NorA protein is coproduced with the respiratory nitric oxide (NO) reductase. NorA contains a diiron-oxo center, which can form stable adducts with dioxygen and NO. In contrast to other diiron proteins, the formation of NorA-NO requires both fully reduced protein and additional electrons. A minor fraction of in vitro NorA-NO represents a paramagnetic dinitrosyl iron complex (DNIC), while the major fraction is attributed to a DNIC of the structure {Fe(NO)(2)}, which shows no electron paramagnetic resonance. The NorA-DNIC may be formed either upon direct reaction of the protein with NO or upon incubation with nitrite due to an intrinsic nitrite reduction activity of NorA that liberates NO. NorA can be purified rapidly as a six histidine-tagged derivative from overproducing cells of Escherichia coli. This chapter describes procedures for the preparation of different redox forms of NorA for the formation of NorA adducts with NO, dioxygen, and azide, as well as for the quantification of NorA-bound NO.